Biologicals and biotechnology
Drugs
How:
- Activate (agonist) or deactivate (antagonist) an enzyme: binding directly to the active site or
through allosteric binding
- Add or remove a metabolite (or analogue)
- Add or remove a protein
- Activate, change or deactivate a biological response (cytokines, steroids, peptides, gases)
- Alter the biological system temporarily or permanently
Where:
Drugs work in on receptors on the surface of the cell. An oral drug goes through your stomach,
intestine, blood and it starts spreading (in circulating system). It has to survive enzymes in mouth,
stomach and intestines. It has to escape from intestine and survive the liver. It goes to the receptor and
survives the kidney until it gets eliminated. For small molecules this is easy, but biology gets bigger
(peptides, antibodies, viruses, bacteria… as drugs).
Small molecules penetrate the cell, so you can have receptors in the cell. If you get bigger, they lose
some of that penetration.
Different types of drugs have a different time of working. It depends on how they come in the body
and how they are eliminated. Some molecules go through you (they will never go into your blood
system) and you will just excrete them again. Antibodies can stay 20-28 days in your body. This cannot
be injected orally, but you deliver them intravenously.
ADME = Administration, Distribution, Metabolism, Excretion
Chemical <-> Biological
Biopharmaceutical = an active drug
substance made by a living organism
or from a chemical reaction involving
biological parts.
By biotherapeutic you never get 100%
of the same molecules, this is different
from chemics. You rely on the cell that
makes the product for you. This is a
long complicated process with
different steps. All these different
steps can affect your final product.
Market
Biotherapeutics are increasingly used to produce drugs.
1
,Human growth hormone
hGH is made in the pituitary gland (=hypophysis → also makes a lot of other
hormones) and released in the blood stream. It has an effect on many cell types
and activates IGF-1 synthesis in the liver. Deficiency or overproduction of hGH
can lead to disease: dwarfism, gigantism and acromegaly among other diseases.
So, if you don’t have the hormone, you don’t grow. If you have it too much, you
grow too much. You can fix this by giving extra hGH.
Sequence and structure
hGH is a protein (191 amino acids). This is the main isoform. But there are multiple versions synthesized
in vivo (at least 5 different isoforms possible). An isoform is a protein variant differencing in sequence
(extra or less amino acids) or post-translation modification. There can be differences in function
between isoform.
Previously: hGH was identified and they knew where it was, but not what it is. They started to purify it
and found that mice grew from it. Many therapeutics begin form extraction → purification → identify.
However, hGH is has a high degree of species specificity. It needs to be human or from some related
primates. You can’t always replace it.
The specificity you get because the surface has a very good match. It has a big surface that interacts
with the receptor → you get specificity. If you extract hGH from dead bodies, you will need 108 bodies
to treat 1 patient for 3 years. This is not sustainable. Also purification of biologicals is never perfect →
batches became contaminated with prions → can cause diseases (ex. Prine disease). So a solution is
making it with biotechnology.
Central dogma
If you want to produce a protein in vivo, you need its genetic
information (DNA sequence).
- Bacteria: You have no RNA processing and very few
proteins get modified. You just need to find DNA
sequence to make a protein.
- Eukaryote: here you have RNA processing and proteins get modified. Reverse transcription of
RNA → DNA but important is reverse transcription of mRNA → cDNA.
hGH has 5 exons with at least 4 isoforms.
First research without biotechnology
You find a source of high level of protein so
you will have a lot of RNA. Acromegalic
tumors have a high level of growth
hormone synthesis. You isolate mRNA and
translate it in vitro (checked for size in gel and for aggregation in anti-serum). The next step is cDNA
synthesis from mRNA and you will have a mixture of molecules present. Then we will do cloning: S1
endonuclease + polymerase, ligation of HindIII adaptor and HindIII-digested plasmid. They synthesized
a precursor protein and additional PTM’s but these are not important for the function. Only disulfide
2
, bonds are important. These cannot be made in bacteria. There are a lot of next steps before it hit the
market (purification on scale, formulation,…).
Today
There is more knowledge, databases and high-throughput methods for discovery. There is cheaper DNA
synthesis and better protein expression and purification tools.
The gene sequence is already known so you search if it has bene characterized or if it is in the genome.
You will then sequence mRNA and obtain cDNA. You can also use mass spectrometry to determine if
and which modifications are present. Then you design a process: decide on host, expression tags,
purification strategy and optimize sequence for the chosen host. You don’t have to clone anymore and
you will just order commercially. You express and optimize (ex. changing a sequence). Each of these
steps affect the final product.
Classic molecular biology
Dependent on identifying suitable sites for restriction with endonucleases.
➢ Case-by-case answers/results
➢ Compromise generally required
➢ Expensive
➢ You need multiple enzymes
The pET system is generated by classic methods and has multiple cloning sites (MCS).
Synthetic biology
Traditional: It is a shift of how you think about biology. You take a complex system and make this simpler
until it breaks and that is your minimum system → minimize complexity to make system tractable (=
top-down approach).
Synthetic: we see if we can construct complex systems by assembling small components (= bottom-up
approach).
SBOL = synthetic biology open language
Open standard for the representation of biological designs. It creates a
common language to represent at an abstract level, the information
contained in the underlying DNA.
- Origin of replication = start of replication of DNA/RNA
- Endonuclease cut site = DNA sequence where an endonuclease (an enzyme that cuts DNA
inside a strand) makes a break in the DNA backbone
- Primer binding site = region of a DNA/RNA sequence where a primer binds to initiate DNA
replication or amplification
- Promotor = DNA sequence that serves as a binding site for RNA polymerase and other
transcription factors to initiate transcription.
- Operator = DNA sequence in a gene's regulatory region where repressor proteins bind to
control gene expression.
- Ribosomal binding site (RBS) = sequence on mRNA where the ribosome binds to initiate
translation
- Coding sequence = part of a gene or mRNA that contains the instructions for making a protein
3
Drugs
How:
- Activate (agonist) or deactivate (antagonist) an enzyme: binding directly to the active site or
through allosteric binding
- Add or remove a metabolite (or analogue)
- Add or remove a protein
- Activate, change or deactivate a biological response (cytokines, steroids, peptides, gases)
- Alter the biological system temporarily or permanently
Where:
Drugs work in on receptors on the surface of the cell. An oral drug goes through your stomach,
intestine, blood and it starts spreading (in circulating system). It has to survive enzymes in mouth,
stomach and intestines. It has to escape from intestine and survive the liver. It goes to the receptor and
survives the kidney until it gets eliminated. For small molecules this is easy, but biology gets bigger
(peptides, antibodies, viruses, bacteria… as drugs).
Small molecules penetrate the cell, so you can have receptors in the cell. If you get bigger, they lose
some of that penetration.
Different types of drugs have a different time of working. It depends on how they come in the body
and how they are eliminated. Some molecules go through you (they will never go into your blood
system) and you will just excrete them again. Antibodies can stay 20-28 days in your body. This cannot
be injected orally, but you deliver them intravenously.
ADME = Administration, Distribution, Metabolism, Excretion
Chemical <-> Biological
Biopharmaceutical = an active drug
substance made by a living organism
or from a chemical reaction involving
biological parts.
By biotherapeutic you never get 100%
of the same molecules, this is different
from chemics. You rely on the cell that
makes the product for you. This is a
long complicated process with
different steps. All these different
steps can affect your final product.
Market
Biotherapeutics are increasingly used to produce drugs.
1
,Human growth hormone
hGH is made in the pituitary gland (=hypophysis → also makes a lot of other
hormones) and released in the blood stream. It has an effect on many cell types
and activates IGF-1 synthesis in the liver. Deficiency or overproduction of hGH
can lead to disease: dwarfism, gigantism and acromegaly among other diseases.
So, if you don’t have the hormone, you don’t grow. If you have it too much, you
grow too much. You can fix this by giving extra hGH.
Sequence and structure
hGH is a protein (191 amino acids). This is the main isoform. But there are multiple versions synthesized
in vivo (at least 5 different isoforms possible). An isoform is a protein variant differencing in sequence
(extra or less amino acids) or post-translation modification. There can be differences in function
between isoform.
Previously: hGH was identified and they knew where it was, but not what it is. They started to purify it
and found that mice grew from it. Many therapeutics begin form extraction → purification → identify.
However, hGH is has a high degree of species specificity. It needs to be human or from some related
primates. You can’t always replace it.
The specificity you get because the surface has a very good match. It has a big surface that interacts
with the receptor → you get specificity. If you extract hGH from dead bodies, you will need 108 bodies
to treat 1 patient for 3 years. This is not sustainable. Also purification of biologicals is never perfect →
batches became contaminated with prions → can cause diseases (ex. Prine disease). So a solution is
making it with biotechnology.
Central dogma
If you want to produce a protein in vivo, you need its genetic
information (DNA sequence).
- Bacteria: You have no RNA processing and very few
proteins get modified. You just need to find DNA
sequence to make a protein.
- Eukaryote: here you have RNA processing and proteins get modified. Reverse transcription of
RNA → DNA but important is reverse transcription of mRNA → cDNA.
hGH has 5 exons with at least 4 isoforms.
First research without biotechnology
You find a source of high level of protein so
you will have a lot of RNA. Acromegalic
tumors have a high level of growth
hormone synthesis. You isolate mRNA and
translate it in vitro (checked for size in gel and for aggregation in anti-serum). The next step is cDNA
synthesis from mRNA and you will have a mixture of molecules present. Then we will do cloning: S1
endonuclease + polymerase, ligation of HindIII adaptor and HindIII-digested plasmid. They synthesized
a precursor protein and additional PTM’s but these are not important for the function. Only disulfide
2
, bonds are important. These cannot be made in bacteria. There are a lot of next steps before it hit the
market (purification on scale, formulation,…).
Today
There is more knowledge, databases and high-throughput methods for discovery. There is cheaper DNA
synthesis and better protein expression and purification tools.
The gene sequence is already known so you search if it has bene characterized or if it is in the genome.
You will then sequence mRNA and obtain cDNA. You can also use mass spectrometry to determine if
and which modifications are present. Then you design a process: decide on host, expression tags,
purification strategy and optimize sequence for the chosen host. You don’t have to clone anymore and
you will just order commercially. You express and optimize (ex. changing a sequence). Each of these
steps affect the final product.
Classic molecular biology
Dependent on identifying suitable sites for restriction with endonucleases.
➢ Case-by-case answers/results
➢ Compromise generally required
➢ Expensive
➢ You need multiple enzymes
The pET system is generated by classic methods and has multiple cloning sites (MCS).
Synthetic biology
Traditional: It is a shift of how you think about biology. You take a complex system and make this simpler
until it breaks and that is your minimum system → minimize complexity to make system tractable (=
top-down approach).
Synthetic: we see if we can construct complex systems by assembling small components (= bottom-up
approach).
SBOL = synthetic biology open language
Open standard for the representation of biological designs. It creates a
common language to represent at an abstract level, the information
contained in the underlying DNA.
- Origin of replication = start of replication of DNA/RNA
- Endonuclease cut site = DNA sequence where an endonuclease (an enzyme that cuts DNA
inside a strand) makes a break in the DNA backbone
- Primer binding site = region of a DNA/RNA sequence where a primer binds to initiate DNA
replication or amplification
- Promotor = DNA sequence that serves as a binding site for RNA polymerase and other
transcription factors to initiate transcription.
- Operator = DNA sequence in a gene's regulatory region where repressor proteins bind to
control gene expression.
- Ribosomal binding site (RBS) = sequence on mRNA where the ribosome binds to initiate
translation
- Coding sequence = part of a gene or mRNA that contains the instructions for making a protein
3
