Biologics and Biopharmaceuticals
(PHEN607)
Faculty of Pharmaceutical Engineering
Faculty of Pharmaceutical Engineering
Biologics and Biopharmaceuticals
(PHEN607)
Dr. Rana A. Youness
Assistant Prof. Molecular Genetics and Biochemistry
Head of Molecular Genetics Research Team (MGRT)
E-mail: rana.youness@giu-uni.de
Office: S1-610
Office Hours: Tuesday 12-1 pm
Biopharmaceuticals Production Technical
Challenges
Lecture 3
Intended Learning
Outcomes (ILOs):
By the end of the lecture and after reading the
appropriate text books, the student should be able to
understand:
• Explore the nature of the vectors used in rDNA
technology
• Determine the main technical problems associated
with rDNA technology and biopharmaceuticals
production
• Analyze the provided solutions for overcoming the
main challenges encountered during
biopharmaceuticals production
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Biopharmaceuticals
3
Health and Disease Applications
(Development of Recombinant hormones-Insulin)
4
QUESTION
How could we modify the inserted
genes in order to facilitate the
purification of the gene product
(protein)?
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Generalised Representation of a Plasmid
Re-Engineered for Heterologous Protein Production
Signal Sequence (Only Purification Tag
if the protein is to be Purification 'Tag'
secreted)
The affinity tags are unique
Promotor Sequence proteins/peptides that are attached at
the N- or C-terminus of the
Promoters are found upstream of Heterologous gene recombinant proteins. These tags help
target genes. They control in protein purification
transcription i.e: needed for the
protein expression Promoter
Sequence
ORI (Origin of
Replication Selection Marker
Sequence) CEN sequence
(FUNGI ONLY)
Antibiotic resistance gene added to
the plasmid to allow easy selection of
transformed bacterial cell as it will
acquire the ability to survive in a
SELECTION MARKER (Ab resistance) medium containing the antibiotic
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DNA Sequence for a Synthetic Gene Constructed
for Expression in a Novel Host
C L E A V A G E
S IT E C L E A V A G E
S IT E
P R O M O T E R
H E T E R O L O G O U S G E N E P U R IF IC A T IO N
S E Q U E N C E
(F R O M D = IF F E R E N T S P E C IE S T A G
The purification tag is only used
during the purification process
then it can be removed to leave a
pure protein
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for transcription needed for protein expression
What you must check in your vector
A. The Promoter:
1- Efficiency: High efficiency or low efficiency
2- Type: Inducible/constitutive
• Inducible: is a regulated promoter that allows
transcription of its associated genes only in
response to specific stimuli
• Constitutive: is an unregulated promoter that
express its associated genes continuously in the
cell
Inducible gene expression systems are more favored than
the constitutive expression system in a wide variety of
basic and applied research areas, including
biopharmaceutical protein production and drug
discovery
This is because they are mostly reversible and thus more
flexible to use. Furthermore, compared to constitutive
expression, they generally exhibit a higher efficiency and
have fewer side effects, such as cell death and delayed
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growth or development
What you must check in your vector
B. Mechanism of vector/plasmid
integration:
Not-integrating plasmid or episomes
Episomal plasmid have higher efficiency but
holds more risks
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Challenges during biopharmaceuticals
production
1- High levels of expression
High levels of expression 30-40% of
cellular protein leads to the production of
inclusion bodies, which consist of protein
and RNA molecules forming an insoluble
complexes
Aggregated proteins tend to eventually
accumulate in host cells as inclusion bodies
which is considered to be a main bottleneck in
the protein production processes and their
formation has been associated to improper rDNA
protein folding or misfolding
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Quick Recap for
Protein Synthesis
11
Challenges during biopharmaceuticals
production
2- Recombinant Protein Degradation
We should consider in-cell or extracellular
degradation of the produced recombinant
protein by the host cell proteases
For example:
Production of recombinant β endorphin, you
should choose strains that are deficient in
one or more proteases are used
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Challenges during biopharmaceuticals
production
3- Incorrect di-sulphide bridging in recombinant protein
Protein Disulphide Isomerase (PDI) provides a
critical role in rearranging proteins with incorrected
disulfide pairings
PDI breaks the disulfide bonds of misfolded proteins
and re-structure it correctly, producing functionally
active recombinant protein
YET, to obtain correct disulfide folding in proteins
Only secretion gives correct -S-S- arrangement, since protein disulfide
isomerase (PDI) is linked to the secretion machinery of the cell
13
Challenges during biopharmaceuticals
production
4- Secretion of Recombinant Protein
To overcome problems associated with the
production of heterologous/recombinant
proteins in E. coli that have structural
disulphide bonds
Use thioredoxin (Trx) fusion proteins
in the vector
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Challenges during biopharmaceuticals
production
Thioredoxin fusions have proved to be especially useful in avoiding inclusion
body formation in the E. coli cytoplasm. E. coli thioredoxin is a compact,
highly soluble, and thermally stable protein with robust folding
characteristics.
These properties perhaps allow the molecule, when fused to a protein of
interest, to serve as a covalently joined molecular chaperon. Thioredoxin
may, thus, act to prevent the aggregation and precipitation of fused
nascent proteins, giving them an extended opportunity to adopt their
correct tertiary folds.
Thioredoxin also possesses several additional characteristics that suits it for
the role as a fusion partner. It is small, highly translated, and its tertiary
structure reveals that both its amino and carboxyl termini are accessible
for potential fusions to other molecules.
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Challenges during biopharmaceuticals
production
5- Glycosylated protein expression
Other problems that may arise regarding
protein expression and reengineering include:
The Heteroprotein e.g: a Glycoprotein
(protein undergoes post-translational
glycosylation)
This causes problems as Glycosylation is
different in animals, plants and fungi and is
not performed in bacterial expression
systems
N.B. the glycosylation is very important for
both the structure and function of the
heteroprotein and the half-life of the
protein/peptide in e.g. blood plasma 16
Challenges during biopharmaceuticals
production
5- Glycosylated protein expression
How to solve this
problem?
Expressing the protein in an organism/cell line
derived from the same kingdom (e.g. Fungi)
This normally gives rise to correct glycosylation of the heterologous protein
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Recombinant Proteins Produced Industrially in Specific
Organisms
Organism/Cell Type Proteins Produced
E.coli Interferon α2a,,α2b, β1b,γ1b; Interleukins 2,11;
Lyme disease vaccine, Insulins
Saccharomyces cerevisiae Vaccines (hepB and C; diptox, pertusis, polio),
anticoagulants, Insulins, glucagon,
BHK cells Blood factors
Chinese hamster Ovary (CHO) cells Interferon β1a, β1b; Blood factors, anticoagulants,
TSH; Gonadotropin, hLeutinising hormone,
Erythropoietins
Mab
Mammalian Cell Lines Erythropoietin, Mab
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