Biotechnology of drugs – Introduction
Doc. RNDr. Jan Hošek, Ph.D.
hosekj@pharm.muni.cz
Department of Molecular Pharmacy
FaF MU
Syllabus of lectures 2024
1. week 19.02.2024 9:00 - 11:30
Introduction to pharmaceutical biotechnology, status, historical milestones; The cell as a biotechnologist's
tool, the function of the endoplasmic reticulum, the Golgi apparatus, post-translational processes,
chaperones
Hošek
2. week 26.02.2024 9:00 - 11:30 Basics of genetic engineering I - principles of how to obtain a recombinant gene, vectors Hošek
3. week 04.03.2024 9:00 - 11:30
Fundamentals of Genetic Engineering II - preparation of genes for cloning, host cells, transformation,
selection and identification of transformants
Hošek
4. week 11.03.2024 9:00 - 11:30 Expression of recombinant proteins in prokaryotic cells and yeast Bartoš
5. week 18.03.2024 9:00 - 11:30
Expression of recombinant proteins in eukaryotic cells - insect cells and baculoviruses, mammalian cells
and adenoviruses
Hošek
6. week 25.03.2024 9:00 - 11:30 Classic biotechnological procedures in pharmacy Bartoš
7. week 01.04.2024
8. week 08.04.2024 9:00 - 11:30
Biotechnological process - definition of the term, stages of the biotechnological process, raw materials for
the biotechnological process, fermenters and bioreactors, methods of product purification
Bartoš
9. week 15.04.2024 9:00 - 11:30 Genetic engineering in higher plants - plant genome structure, vectors, expression cassettes Bartoš
10. week 22.04.2024 9:00 - 11:30 Plant biotechnology - methods of transformation and identification of transgenic plants, molecular farming Bartoš
11. week 29.04.2024 9:00 - 11:30
Examples of classical and recombinant biotechnology products in pharmaceuticals, cytokines as tools in
the fight against infections and in tumor therapy, hormones, enzymes, antibodies and their derivatives
Hošek
12. week 06.05.2024 9:00 - 11:30 Gene and cell therapy, tissue engineering, genomics and proteomics in pharmacy Hošek
13. week 13.05.2024 9:00 - 11:30
Application of genetic engineering and plant biotechnology in pharmacy, medicine and food industry,
issues of dealing with genetically modified organisms
Bartoš
14. week 20.05.2024 9:00 - 11:30 Preparation of recombinant vaccines
Biology
(1st year)
Microbiology
(2nd year)
Molecular
biology
(2nd year)
Biochemistry
(2nd year)
Patobiochemistry
(2nd year)
Methods of
molecular
biology
(3rd year)
Biotechnology
of drugs
(3rd year)
Biotechnology
of drugs -
practical
classes
(3rd year)
Knowledge-flow of biology subjects on DMP
Recommended literature
➢ Essential Cell Biology – Alberts et al., 1998
➢ Pharmaceutical biotechnology – Crommelin et al. 2013
➢ Biotechnology Foundations, 2nd Edition – Jack O´Grady, 2019
(https://legacy.cnx.org/content/col26095/1.5)
What is BIOTECHNOLOGY?
Processes using living organisms
or their components to produce or
modify products; breeding of
animals, plants and microorganisms
for specific uses (commercial
application and benefit to mankind).
Biotechnology
Molecular
biology
Genetics Microbiology Biochemistry Chemistry Bioengineering Enzymology
Prof. Drobník
The nature of biotechnology is intrinsically linked to the
application of biology, i.e
with industry, and thus also with commerce..
Therefore, it is necessary to operate it in such a way that people
benefit from its results as much as possible, which is not
possible without a commercial outcome.
For that reason, the quality of scientific work in biotechnology
should be measured by patents rather than the impact factor of
publications.
Biotechnological outputs
• Food and beverage production
• Production of materials and fuels
• Production of drugs and medical materials
Biopharmaceuticals
Biopharmaceuticals are drugs or potential drugs obtained by other
than the classical route of synthetic chemistry (including combinatorial
chemistry) or drugs originally obtained by isolation from plant
(biological) material.(Beneš, 2007, Chem. Listy 101, 18−24)
• Creation using genetic engineering and recombinant technologies
• Therapeutic proteins, DNA or RNA, antibodies, parts or whole cells,
drug conjugates with proteins or natural polymers,...
Classification of biotechnology
• Classic microbial technology
• Enzyme technologies
• Use of animal and plant cells
• Gene technology
• Pharmaceutical biotechnology
Pharmaceutical biotechnology
According to European Association of Pharma Biotechnology (EAPB)
…is the science that covers all the technologies necessary
to create, manufacture and register biotechnological drugs.
Is it really science?
1) Existence of the subject of research
2) Central theory
3) Existence of methodological approaches
4) Institutionalization
5) Own journal
Subject of pharmaceutical biotechnology
• The subject of studying pharmaceutical biotechnology is medicine
created by a specific (biotechnological) procedure - using
organisms, living cells or their components.
• It requires a specific approach in all stages of production - own
design, development and finally production.
• Medicines created through biotechnological procedures are subject to
a special registration regime, as they are products of genetically
modified organisms.
Central theory of pharmaceutical biotechnology
➢ common evolutionary origin of organisms
➢ the universality of the genetic code across living systems
➢ similarity of transcription and translation apparatuses
Substances biologically active in one organism can be
produced in any other organism..
1) The recombinant product (although not produced in its
original organism) is functionally identical or at least very
similar to the "original".
2) A biopharmaceutical product can be appropriately
modified by a biotechnologist to have special properties
that allow it to perform a specific function in the target
organism.
Methodical approach of pharmaceutical biotechnology
Diversity of sources = wide spectrum of methods
• Drug development = molecular biology, genetics and genetic engineering
• Purification and characterization of products = biochemistry, analytical
chemistry
• Drug Development = Biochemistry, Enzyme Engineering, Organic
Chemistry, Pharmaceutical Chemistry, Drug Technology and
Pharmacology
• Drug registration = applied pharmacy
• Drug production = classic microbiology, genetics
The specifics of the methodical approach
COMPLEXITY
Example of recombinant protein production
Gene isolation
Cloning into
production cells
Clone
characterization
Recombinant
protein production
Product separation
and purification
Characterization
and chemical
definition
Clinical trials
Administrative
registration
process
Postclinical trials
Specific methodological approaches
• Gene therapy
• DNA/RNA vaccines
• siRNA-based drugs
DOI:10.1038/aps.2017.2
doi.org/10.1016/j.nano.2020.102239
Scientific societies
The European Association of Pharma Biotechnology, EAPB
http://www.eapb.org/
The American Association of Pharmaceutical Scientists (AAPS)
https://www.aaps.org/home
Research institutions
• Foreign:
– The North Carolina Central University, Biotechnology and
Pharmaceutical Law Institute
(https://www.nccu.edu/research/institutes-and-special-
facilities/bpli)
– Department of Pharmaceutical Technology and
Biopharmaceutics, Martin-Luther-University Halle,
Germany (https://www.pharmazie.uni-
halle.de/institutsbereiche/)
• Czech:
– PRO.MED.CS Praha a.s. (https://www.promed.cz/)
– Biopharm – Výzkumný ústav biofarmacie a veterinárních
léčiv, a.s., Jílové u Prahy, Česká republika
(https://www.bri.cz/)
Journals
• According to WoS, 170 journals in the BIOTECHNOLOGY &
APPLIED MICROBIOLOGY category (February 2024)
• Current Pharmaceutical Biotechnology
(https://benthamscience.com/journals/current-
pharmaceutical-biotechnology/)
A brief history of biotechnology
• Initial period to 1850
• The Era of Pasteur
• The era of industrial biotechnology
• The era of new biotechnologies
• Transgenic organisms
Biotechnology in prehistoric times…
• empirical use of wild microorganisms to prepare
fermented products
• in Mesopotamia by the Sumerians in the 7th
millennium BC – beer production
• about 4,000 years BC the use of yeast to ferment
bread dough in ancient Egypt
• about 4,000 years BC preservation of milk by
fermentation in ancient China – production
of cheese, wine, vinegar
The beginnings of pharmaceutical biotechnology
• The discovery of vaccination
– Edward Jenner 1796 – first vaccination against smallpox
https://commons.wikimedia.org/wiki/File:Edward_Jenner-_Smallpox.svg
Difficult beginnings of vaccination…
Engraving by
Jamese Gillraye
(1802)
The Era of Pasteur
• Louise Pasteur (1822 – 1895) - assumes
that fermentation is caused by living
microorganisms; discovery of heat
sterilization - pasteurization; vaccine
research based on killed pathogens
(anthrax, rabies)
• Eduard Buchner (1860 – 1917) –
demonstrated fermentation using yeast
cell-free extract - basics of enzymology
The era of industrial biotechnology
• Chaim Weizmann (1874 – 1952) – the first
industrial production of acetone from
corn starch using a pure culture of
Clostridium acetobutylicum
• Alexander Fleming (1881 – 1955) –
discovery of the fungus Penicillinum
notatum and its antibacterial activity (1928)
– Howard Florey, Ernst Boris Chain and
Norman Heatley (1940) – description of the
isolation of pure penicillin and its industrial
production – the first biotechnological
product
J.Tramper,Y.Zhu,Modern
Biotechnology–Panaceaornew
Pandora’sbox?
The era of new biotechnologies I.
• 70s‘ – discovery of restriction endonucleases  creation of
recombinant DNA  basics of genetic engineering (Paul Berg,
Herbert W. Boyer, Stanley N. Cohen)
• 1972/3 – transfer of foreign DNA into a bacterium  the first
transgenic organism
• recombinant technology
– targeted genetic modification
information
– GMOs
– production of proteins in modified
form
https://www.sciencedirect.com/topics/chemical-engineering/genetic-engineering
The era of new biotechnologies II.
• 1980 - Cohen and Boyer obtain
the first US patent for cloning
and creation of genetically
modified microorganisms
• 1982 - The FDA approved the
first drug produced by
biotechnology techniques, a
human insulin, Humulin®,
produced by genetically
modified bacteria
Human Insulin: From Gene to Drug Human insulin chains are made
by recombinant DNA technology and then combined to produce the
widely used drug.
The era of new
biotechnologies III.
• 1975 – discovery of hybridomas (César Milstein
and Georges J. F. Köhler) – cells created by the
fusion of B-cells and myeloid cells  mass
production of monoclonal antibodies
© Brandlee86~commonswiki
© Biological Industries
Transgenic organisms
• Development since the 1970s (1972 – viruses,
1973 – bacteria, 1974 – claw, 1974 – mouse,
1983 – tobacco, 1985 – cow, 2000 – rice)
• Animals
– production of human proteins (e.g. anticoagulant
protein in goat milk)
– disease models (knock-out and knock-in organisms)
• Plants
– more resistant to herbicides, immune to harmful
insects; modified appearance (bigger fruits, flowers)…
– higher production of biologically active substances
(e.g. vitamin A in rice)
doi:10.1186/1471-2407-12-21
https://www.flickr.com/photos/ricephotos/5516789000/in/
set-72157626241604366
Modern milestones
• 1978 Human insulin is produced for the first time
• 1980 US Supreme Court - patent protection of a biotech product
• 1982 The FDA approved the first biotechnologically prepared
drug – human insulin
• 1983 PCR technique discovered
• 1986 Recombinant human hepatitis B vaccine
The first field tests of a transgenic plant – tobacco
• 1997 The first animal cloned from an adult cell - Dolly the sheep
• 2004 FDA approves first anti-angiogenic cancer drug,
Avastin (bevacizumab)
Industrial application of biotechnology
Biotechnology in industry means
→ appropriate addition of chemical technology
→ expanding the range of manufacturable
substances
• Substances that can only be produced by
chemical synthesis
• Substances that can only be produced
using biotechnology
• Substances that can be produced by
chemical technology and biotechnology
Advantages and disadvantages of biotechnology
Advantages
• Cheap raw material base
• Lower energy consumption
• Higher speed of biochemical
events
Disadvantages
• High R&D and initial
investment costs
• Little efficiency
• Low concentration of
the substances
involved
• Still unclear risks
Considerations in choosing between
synthesis and biotechnological manufacturing
• technical feasibility of the biotechnological process
• current price of raw materials
• ease of isolation of the final product and its purity
• the possibility of obtaining useful by-products
• success in terms of speed of commercialization, etc.
Main areas of use of biotechnology in the
pharmaceutical industry
• Natural metabolites
• Biosynthesis; all the information
needed for such production can be
found in the genome of the cell
• e.g. ATB, vitamines etc.
• Substances prepared by
biocatalysis
• Biotransformation
• e.g. steroid compounds
• Human proteins and polypeptides
• Gene manipulation
21st Century Guidebook to Fungi, SECOND EDITION
DOI: 10.1186/1475-2859-12-95
Examples of the use of biotechnology in
medicine and pharmacy
• Amino acids, vitamins (riboflavin B2, pyridoxine B6, vitamin B12, biotin H,
β-carotene, astaxanthin, etc.), polysaccharides (dextran, xanthans,
pullulan, etc.)
• Medicines (antibiotics, steroids, antifungals, ergot alkaloids...)
• Monoclonal antibodies, vaccines
• Interferons, interleukins
• Hormones - human growth hormone, FSH, insulin
• Production enzymes and pure enzymes for bioanalytical methods
(immunoassay)
• Gene therapy
Biotechnology of drugs – Proteins,
major biopharmaceutical
Tertiary structure of proteins
It is crucial for the creation of functional molecules –
thermodynamically optimal conformation
It includes the following processes:
➢ Creation of 3D conformation
➢ Addition of cofactors
➢ Modification by kinases or other
protein-modifying enzymes
https://doi.org/10.3389/fnins.2020.611285
Forces responsible for the 3D structure of proteins
Primarily non-covalent interactions
Ion binding Hydrogen
bridge
van der Waals force
Hydrophobic interactions
… is the most important for 3D creation
Overview of steps in 3D protein formation
nascent polypeptide
chain
Folding and cofactor binding
(non-covalent interaction)
Glycosylation, phosphorylation,
acetylation (covalent interaction)
Binding to others
protein subunits
mature functional
protein
PP
OpenStax College - Anatomy & Physiology, Connexions Web site.
http://cnx.org/content/col11496/1.6/
Why is knowledge of the formation of 3D
protein structures important for a
pharmacist?
Understanding the very structure of proteins and
the processes of its formation is important for
drug design, because drugs often act at the level
of the binding or allosteric site of enzymes.
Where to study protein structures?
Protein Sequence Database
SWISS-PROT database established at the University of
Geneva in 1986
Managed by the Swiss Institute for Bioinformatics(SIB)
Database PDB (The Protein Databank)
It archives and analyzes protein structures and complexes of informative
biomacromolecules
Contains automatically completed translations of gene sequences
from EMBL (www.ebi.ac.uk)
http://www.rcsb.org/pdb/home/home.do
www.expasy.org
➢ an autonomous process requiring no additional
factors or energy input
➢ Christian Anfinsen 1972 Nobel Prize
➢ tertiary structure after leaving the ribosome in vivo
➢ native conformation until the moment of
degradation
➢ chaperones help protein folding
Chaperones a chaperonines
• chaperones (monomers 70 – 100 kDa) are proteins that help the
covalent folding or unfolding and assembly or disassembly of other
macromolecular structures (mainly Heat shock proteins – Hsp)
• chaperonines (oligomers 800 kDa) are a class of molecular
chaperones that provide favorable conditions for the correct folding of
denatured proteins, thus preventing aggregation (e.g. GroEL, TRiC)
• they do not provide steric information
• they inhibit unproductive interactions
• they are located in all compartments
• protein packaging
• conformational rearrangements
https://doi.org/10.1016/S0960-9822(99)80082-7
Chemical chaperones
• Small molecules that bind to misfolded proteins and
stabilize them or ensure their proper folding
• They stabilize poorly packed proteins, reduce
aggregation, prevent unproductive interaction with
other proteins
• E.g. glycerol, trimethylamine N-oxide
• Re-aggregation of inclusion bodies upon
expression of recombinant proteins
It is used, for example, for treatment of
➢ Amyloidosis transthyretin (Tafamidis)
➢ Cystic fibrosis
➢ and other diseases associated with protein
packaging
https://doi.org/10.1203/00006450-200212000-00004
The result of folding = creation of an active
conformation
Alberts et al 2008: Figure 3-37a Molecular Biology of the Cell (© Garland Science 2008)
Formation of disulfide bridges in the cell
• Part of the protein folding process
• It takes place in the periplasmic space of
prokaryotes and in the mitochondria and
ER of eukaryotes
J. Riemer et al., Science 324, 1284 -1287 (2009)
Eukaryota
Post-translational modification of proteins
Some processes are co-translational, others truly
post-translational
The most common is glycosylation
Modification changes the structure and
biological activity of proteins
Other post-translational modifications of proteins
➢ Most often glycosylation
or phosphorylation
➢ In total, more than 300
types of covalent
modifications of
polypeptide chains have
been described
Modification of therapeutic proteins
Modification type Importance
Glycosylation
 solubility, affects biological half-life or biological
activity
Carboxylation
Important for the binding of some blood proteins to
Ca2+
Hydroxylation Important for structural arrangement
Sulphatation
It affects the biological activity of neuropeptides
and the proteolytic processing of polypeptides
Amidation It affects biological activity and stability
Glycosylation
➢ attachment of a sugar residue to a polypeptide chain
➢ it occurs mainly in extracellular proteins and proteins found on the surface of cells
Glycosylation produces glycoproteins
➢ For some proteins, deglycosylation has no effect on biological activity
➢ Deglycosylated forms can be prepared by the action of glycosylation inhibitors,
e.g. the antibiotic tunicamycin in the growth medium or by enzymatic degradation
of the glycidic part of the preformed glycoprotein with glycosidase.
➢ Differential glycosylation causes problems in heterologous expression
systems
Effects of the glycidic part of glycoproteins
➢ Protein packaging
➢ Protein transport/targeting
➢ Ligand recognition/binding
➢ Biological activity
➢ Stability
➢ It regulates the biological half-life of the protein
➢ Immunogenicity
Human gonadotropic hormone
➢ Highly glycosidated
➢ Removal of the glycidic moiety
results in loss of biological
activity, although the hormone
still binds to its receptor,
sometimes even with higher
affinity
Cell – a pharmaceutical biotechnologist's tool
a small, membrane-bound unit filled with a concentrated aqueous
solution of chemical compounds and endowed with the
extraordinary ability to make copies of itself by growth and
division
Cell meaning
• The cell serves as a source of starting materials, because only the cell is
capable of creating new compounds that are useful as medicine.
• Except in very exceptional situations, when we use only some of its parts for
so-called in vitro translation, the cell is the factory where all
biotechnological processes take place.
Prokaryotes vs. eukaryotes
What interests the biotechnologist about the cell
• Everything related to protein formation and localization
• Genophore
• Endoplasmic reticulum
• Golgi apparatus
• Rough ER
– is studded with ribosomes
– is the site of protein formation
• Smooth ER
– Opening vesicles that carry newly
formed protein and lipid molecules
for intracellular transport
– It forms a network of thin channels
connected directly to the rough
plasmatic reticulum
– Enzymes catalyzing the
transformation of lipids are bound
to its membranes; in specialized
cells also steroid hormones,
arising from cholesterol
Multiple curved membrane sheet,
which forms closed sac - endoplasmic
lumen (cisternal space)
https://micro.magnet.fsu.edu/cells/endoplasmicreticulum/endoplasmicreticulum.html
➢ central role in the synthesis of lipids, proteins, steroids
➢ it facilitates the formation of the correct tertiary or quaternary
structure of proteins
➢ transport system – distribution of proteins to the cytoplasm or
organelles
➢ maintaining osmotic pressure
➢ chemical modification of proteins
▪ disulfide bridges are formed by oxidation of cysteine ​​pairs of side
chains
▪ formation of glycoproteins by covalent attachment of a short
oligosaccharide side chain – is completed in GA
▪ the oligosaccharide precursor is linked by an O- or N-bond to a protein
molecule
▪ protein output is controlled = misfolded protein is retained by
chaperone or degraded
Detoxification enzymes
Ca2+
Ca2+
cholesterol
steroid
hormones
toxin - lipid
toxin - water
COO-
COO-
NH2
NH2
transmembrane
protein
misfolded
enzymes of
steroid synthesis
Components of
lipids synthesis
orientation
lumen ER
ER membrane
soluble protein
Glykosylation
S-S bridges
chaperones
SRP Sec61 translocator, SRP receptor
growing bodies
transport to GA
Schematic representation of ER functions
DOI: 10.1016/j.biocel.2011.10.012
Golgi apparatus (GA)
• modification and sorting of
proteins and lipids for secretion or
for delivery to another organelle
Described in 1898 by the Italian
cytologist Camillo Golgi in nerve cellshttps://www.sciencefacts.net/golgi-apparatus.html
Golgi apparatus (GA)
• Complex of cisternae and vesicles
• Located close to nukleus and ER
• Vesicles – sacs containing proteins
produced in the rough ER deliver their
contents to the cis face of the GA with
which they fuse
• Secretory vesicles – vesicles containing
processed proteins and suffocating from
the trans face of the GA from where they
move to the plasma membrane where
they excrete their contents into the
extracellular environment
https://www.sciencefacts.net/golgi-apparatus.html
GA functions
• Transport and storage of differnt
compounds
• Posttranslation modification of proteins
– The most common are
glycosylation, phosphorylation,
sulphatation, specific proteolysis
• Synthesis of polysaccharides and
immunoglobulins
• Creation of secretory vesicles used during
exocytosis
• Production of materiál for cell wall
• Creation and diferentiation of lysosomes
• Reparation of cell surface
• Creation of vacuols
© 2009 Nature Publishing Group Xu, D. & Esko, J. D. A Golgi-on-achip
for glycan synthesis. Nature Chemical Biology 5, 612–613 (2009)
For the pharmaceutical
biotechnologist, they are
sources of genetic information
and tools for gene manipulation
Genophores
• Nuclear chromosomes
• Mitochondrial and chloroplast DNA
• Bacterial nucleoid
• Plasmids
Cell
structure
Metabolic event
nucleus DNA biosynthesis, RNA biosynthesis and modification
cytoplasm glycolysis, pentose cycle, biosynthesis of carbohydrates and fatty acids
mitochondria
respiratory chain and oxidative phosphorylation, citrate cycle, fatty acid
degradation, aminoacid metabolism
ribosomes protein biosynthesis
Endoplasmic
reticulum
synthesis, modification and transport of some proteins, synthesis of
cholesterol, phospholipids and triacylglycerols, detoxification
Golgi apparatus modification, sorting, transport and excretion of some proteins
lysosomes breaking down worn-out biomacromolecules and foreign structures
peroxisomes oxidation to form hydrogen peroxide, photorespiration
chloroplasts photosynthesis, fatty acid synthesis
glyoxisomes glyoxylate cycle