University of Indonesia / Jakarta

March 6th 2019

Biotechnology in Drug Development

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

Biotechnology in Drug Development

1. INTRODUCTION

What is Pharmaceutical Biotechnology, history, market potential of

pharmaceuticals

2. MAIN PART

Basics of Pharmaceutical Biotechnology

– Medical needs
– Target identification / validation
– Compound libraries
– Assay establishment and agent screening
– Lead optimization
– Recombinant production of pharmaceutical proteins
– Safety Aspects
 Recombinant Drugs

– In USA and Germany more than 200 approved recombinant

drugs (in Germany 272 containing 221 different agents)

– Part of recombinant drugs in the entire world market increased

from 1985 by factor 20, from 1995 by factor 4.
 Recombinant Drugs

– Hormones (e.g. Insulin),

– Growthfactors / Immunomodulators (e.g. Erythropoetin and Interferones),
– Enzymes (e.g. Tissue Plasminogen Activator),
– Inhibitors (e.g. Hirudin)
– Antibodies (e.g. Herceptin) and
– Vaccines (e.g.Hepatitis-B-Antigen)
Recombinant production of proteins (drugs)
Basic principle of recombinant protein production
Nowadays, the required DNA fragment may be ordered from a company
 Reasons for recombinant production of
peptides/proteins

– Production of different factors for basic research (analysis of

structure/function, production of frequently used factors such as
enzymes etc.)

– Large scale production of (sometimes optimized) industrial

enzymes

– Production of enzymes and antibodies used in diagnostics

– Production of pharmaceuticals (hormones, enzymes, vaccines

etc.)
 Advantages of recombinant production

– Yield (upscaling is possible), otherwise not available

– Harmlessness (e.g. no virus contamination)

– Production of endogenous human substances is possible

(less side effects)
Basic principle of recombinant protein production
 Posttranslational modifications of peptides/proteins
in eukaryotes

Modification Function / effect

– Generation of disulfide bonds Stabilization of structure

– Glycosylation Folding, biological function,
solubility, stability, secretion,
dwell time in blood,
antigenicity (!!!)

– Proteolysis Removal of signal peptides,

– C-terminal amidation
– Phosphorylation
– Many more (e.g. acetylation,
carboxylation, sulfation....)
activation (inactivation)
Stabilization of peptides
Activation / inactivation
Examples for frequent N-glycosylations in
different organisms
 Important aspects concerning the
quality of the product

1. Selection of a suitable organism–> the more complex the

degree of required posttranslational modifications the
higher the required organism

2. Selection of a suitable cell compartiment –> in E.coli

periplasmatic space for generation of disulfide bonds

3. Cotransfection with chaperone genes („helpers“ for correct

folding) or genes for required modifying enzymes (e.g.
glycosyltransferases)
 Important aspects concerning the yield of the
peptide / protein to be produced

1. Gene dosage Copy number of the expression vector,

number of copies of the gene to be integrated in the
genome

2. Transcription Gene dosage and promoter strength

3. Translation mRNA generation (transcription) and stability (3'-

terminus), Shine-Delgarno or Kozak-sequence,
codon-Usage

4. Stabilty Posttranslational modifications, proteinase deficiency

of the production strains
Criteria for selection of a suitable
expression system
Organisms / systems used for recombinant
production of peptides / proteins
Basic principle of recombinant protein production
 Typical components of an expression vector

– Origin of replication (ori)

– Selective marker (often antibiotics resistance)

– Strong inducible promoter

– MCS, enabling an "in frame" - cloning of the gene / cDNA to be

expressed; nowadays also T-cloning

– Certain fusion parts enabling an easy detection and purification of the

product (e.g. His-Tag – usually 6 Histidin residues)

– Endoproteinase cleavage site enabling the separation of the fusion part

from the desired product

– Terminator of transcription
Inducible Promoters in E.coli Expression Systems
Arabinose-Inducible Promoter
 Typical components of an expression vector

– Origin of replication (ori)

– Selective marker (often antibiotics resistance)

– Strong inducible promoter

– MCS, enabling an "in frame" - cloning of the gene / cDNA to be

expressed; nowadays also T-cloning

– Certain fusion parts enabling an easy detection and purification of the

product (e.g. His-Tag – usually 6 Histidin residues)

– Endoproteinase cleavage site enabling the separation of the fusion part

from the desired product

– Terminator of transcription
His-tagged recombinant proteins may easily be purified
by means of Ni-chelate chromatography
Most simple expression organism: E. coli

But: not able to posttranslationally modify

the protein - not even disulfide bonding
Example for an E. coli - expression vector
What can I do, when the protein to be pro-
duced is relatively simple but the functional
form requires disulfide bond(s)?
Solution: „expression“ within the periplas-
matic space of E.coli

More precise: by means of certain signal

sequences (e.g. gIII) the expression product
is directed into the periplasmatic space
Vector for periplasmatic „expression“
Sometimes eukaryotic proteins expressed in pro-
karyotic cells are incorrectly folded (especially if not
directed to the periplasmatic space) and form un-
soluble „inclusion bodies“

These inclusion bodies contain high amounts of

protein, enable easy purification (mainly by density
gradient centrifugation) but are difficult to resolve
Inclusion bodies (containing incorrectly folded protein)

Advantage:
easy to purify

Disadvantage:
difficult to resolve

Generation of unsoluble „inclusion bodies“ with urodilatin nonamer

in the cytoplasm (!) of E. coli 5h after induction
 Yeast Pichia (pastoris or methanolica)

– Still relative easy to handle

– Disulfide bonds are generated

– Glycosylation, but different from mammals

– Desired peptide / protein may be produced without any

fusion part and secreted into the medium
Integration of the Pichia vector into the Pichia genome
 The baculovirus system

– Host cells Sf9 of the butterfly Spodoptera

frugiperda
– High yields but glycosylation different from
mammals
 Spodoptera frugiperda Sf 9 cells

– Expression system based on the Spodoptera-infecting

baculovirus

– Suitable for recombinant production of proteins exhibiting

posttranslational modifications (e.g. glycosylations,
phosphorylations, acetylations)

– The eukaryotic Spodoptera cells may accomplish all of these

modifications
 Spodoptera frugiperda Sf 9 cells

– High expression yields of 1-500 mg recombinant protein per

liter of culture

– Cultivation in T-flasks, spinner flasks or roller cultures

– Disadvantage: mammalian proteins may exhibit differing

posttranslational modifications, e.g. incorrect sugar residues
(of importance for function and allergenic potential)
 Baculovirus expression system

– Based on two components, e.g.

Virus genome with partially deleted gene for the

protein 1629 which is essential for virus reproduction

„Transfer vector “ for cloning of the cDNA for desired

protein behind the strong polyhedrin promoter

– Transfer vector contains missing parts of the gene for protein 1629

– Cotransfection of Sf9 cells with virus genome and transfer vector

– Thereby in parts homologous recombinantion leading to completion

of the gene for protein 1629
The Bac-N-Blue expression system
Baculovirus reproduction
 Expression in mammalian cells
– Proteins undergo correct posttranslation modifications

– Quality of peptides / proteins is high, but cultivation is complex

and expensive

– Transient (temporary) or stable transfection

– Vector may be integrated into the host genome in multiple

copies, selection may be achieved via concentration of
antibiotic, e.g. G418

– CHO are well characterized and suitable

Expression of the cDNA for GFP in CHO cells

Transfection
Also inducible expression systems for
mammalian cells, e.g. T-Rex
Prof. Wolfgang Hillen
Tetracycline-inducible expression system
Tetracycline-inducible expression system
 „Gene Pharming“

– Use of transgenic animals (mainly sheep, cow, pig) for production of

recombinant proteins

– Pharmaceutical important proteins are produced in tissue of lactiferous

glands - purification from milk

– Correct posttanslational modifications (high quality)

– High yields of 100 g up to 1 kg protein per animal and day

– e.g. a herd of 20 - 30 sheeps would be sufficient for production of the

complete amount of blood clotting factor VIII required in the USA

– Disadvantes: complex and difficult techniques required for establishment

of a herd of transgenic animals
Gene pharming
Downstream Processing
 Synthetic Biology

– The design and fabrication of biological components and systems

that do not already exist in the natural world

– The re-design and fabrication of existing biological systems

– More extensive and complex change of an organism compared to

classical genetic engineering – even generation of a novel organism
possible

– Availability of libraries with well-defined genetic building blocks

(„biobricks“)

– Use of these building blocks for generation of new organisms

producing or doing whatever you want
 DNA Synthesizer

Synthesized
genetic
building blocks

Nucleus

Cell Mitochondria

Products
 Use of synthetic biology in pharmaceutical
biotechnology

Some examples:

– Creating in vivo compound libraries

- Advanced and complex metabolic engineering
- Generation of organisms capable of incorporating unnatural
amino acids into a synthesized protein

– Creating specific screening systems with sensitive reporter systems

for the discovery of new drug candidates

– Optimization of the synthesis of pharmaceuticals

– Optimization of drugs (e.g. stability, activity)

 Polyketide Libraries

– Polyketides represent a large family of natural agents with different

medically useful activities, such as antibiotic (tetracyclines, erythromycin),
anti-cancer (doxorubicin, epothilone), and antiparasitic activities

– They are synthesized by polyketide synthases which represent modular

assembly lines

– These modules are ideal for rearrangements (even using mixed modules
from different organisms) leading to hybrid enzymes synthesizing novel, not
naturally occuring polyketides

– First success in synthesizing polyketides in E.coli

Polyketide Synthesis
 Organisms Producing Proteins with Unnatural Amino
Acids

– Generation of genes „encoding“ not naturally occuring tRNAs with STOP

anticodons (e.g. Amber anticodon)

– Modification of genes encoding aminoacyl-tRNA synthetases (aaRS),

selection of such clones synthesizing aaRS capable of recognizing the new
tRNA and a selected unnatural amino acid (UAA)

– UAA is added to the culture medium, transported into the cell, enzymatically
(aaRS) connected to the new tRNA, and incorporated into synthesized
protein(s)

– Increase of chemical diversity of the cellular proteins

– Screening for desired activities

 Optimization of the Synthesis of Pharmaceuticals

Example: Multiplex Automated Genome Engineering (MAGE)

– Feeding cells with synthetic oligonucleotides used as Okazaki fragments

during replication – directed mutations

– Fast generation of multiple different clones

– Screening for clones showing high production rate

– Successfully used by the group of Georg M. Church to mutate and fine-tune

ribosomal binding sites genome-wide resulting in fivefold production of
lycopene in an E.coli strain within only three days of evolution
 Optimization of Drugs

Example: Stabilization by introducing redox-insensitive cross-links

– Protein structures often stabilized by disulfide bridges

– In recombinant proteins disulfide bridges are difficult to reproduce because of

their redox sensitivity

– The group of Heinz Neumann succeeded in incorporating two unnatural

amino acids with an azide and an alkyne function, respectively, in their side
chains into the same recombinant protein, leading to formation of a redox-
insensitive cross-link by a Cu(I) catalyst

– => Stabilization of the protein

Thank you very much for your attention!