Bio Tech products
(Bio Pharmaceuticals),
(Genetically Modified Products)
Ravi Samaraweera,
Dip In Pharmacy
Overview
1. History
2. Manufacturing, Quality control and
safety aspects
3. Types of Bio Pharmaceuticals and
their functions.
4. Practical usage
5. Latest developments
6. Future Developments
• Plants were the origin of western medicine
• Healers used an extract of the leaves or
bark of certain willow species to treat
rheumatism, fever and pain.
• Hundreds of years before( in 1897) the
Bayer chemist Felix Hoffmann reacted the
salicylate in the extract with acetic acid to
form acetylsalicylic acid, a compound that
is gentler on the stomach.
History of western medicine
The term ‘biotechnology’ was first
used in a 1919 by Karl Ereky, a
Hungarian engineer and economist.
He described that new techniques
would provide adequate food for the
rapidly growing world population
• The first medicine to be produced in this
way was the hormone insulin: in the late
1970s Genentech, an American company,
developed a technique for producing
human insulin in bacteria cells and
licensed the technique to the
pharmaceutical company Eli Lilly.
• Until 1982 insulin was isolated from the
pancreas of slaughtered animals via a
complex and expensive process up to 100
pig pancreases being required per diabetic
patients per year.
• However, some patients treated with it
develop dangerous allergic reactions.
• Some 200 million diabetics worldwide now
benefit from the production of human
insulin.
• Without gene technology and
biotechnology this would be impossible: in
order to meet current demands using
pancreatic extract, around 20 billion pigs
would have to be slaughtered annually.
Biotech Products
1. Insulins
2. Interferons
3. Vaccines
4. Growth hormones
5. Products for fertility
6. Erythropoietin
7. Growth Factors
8. Interleukins
9. TNF alfa Blockers
10. Monoclonal antibodies
What are genetically modified (GM)
organisms ?
• Genetically modified organisms (GMOs) can be
defined as organisms in which the genetic
material (DNA) has been altered in a way that
does not occur naturally.
• The technology is often called “modern
biotechnology” or “gene technology”, sometimes
also “recombinant DNA technology” or “genetic
engineering”.
• It allows selected individual genes to be
transferred from one organism into another, also
between non-related species.
Gene
• This establishes a new cell line, which is
usually treated as a closely guarded
company secret.
• After all, these cells are the actual
factories of the biopharmaceutical
concerned.
• They are allowed to reproduce and are
then safely stored at low temperatures in
what is known as a master cell bank.
• The production process is divided into the
following steps:
3. Cultivation:
• The length of this step depends on the type of cell used.
• Under favorable conditions bacterial cells such as Escherichia coli usually divide once every 20 minutes; thus one cell gives rise to 4.7 x 1021 cells within 24 hours.
• By contrast, mammalian cells such as CHO cells divide about once every 24 hours.
• During the growth phase the cell culture is transferred to progressively larger culture vessels.
3. Fermentation
(Along a seed train the culture volume is expanded from ml‘s to thousands
of liters and the cells are secreeting the product into the medium)
Biotech products manufacturing
• Fermentation:
• The actual production of the biopharmaceutical occurs during this phase.
• The culture medium contains substances needed for the synthesis of the desired therapeutic protein.
• In total, the medium contains around 80 different constituents at this stage, although manufacturers never disclose the exact composition.
• The industrial-scale steel vessels in which fermentation takes place have capacities of 10,000 liters or more.
•
• 4.Purification:
• In the simplest case the cultured cells will have secreted the product into the ambient solution. In this case the cells are separated from the culture medium, e.g. by centrifugation or filtration, and the desired product is then isolated via several purification steps.
• If, on the other hand, the product remains in the cells following biosynthesis, the cells are first isolated and digested (i.e. destroyed), and the cellular debris is then separated from the solution together with the product.
• The yield from bioproduction processes is usually much lower than from chemical synthesis.
• For example, a 10,000-liter fermenter yields only a few kilograms of a therapeutic antibody.
• The production steps, including purification, take several weeks. Several more weeks are then needed to test the product.
• Each product batch is tested for purity to avoid quality fluctuations, and a 99.9 percent purity level is required for regulatory approval. Only then can the finished product be further processed and shipped.
• Nowadays all the steps in the production
of biopharmaceuticals are fully automated.
Production staff step in only if problems
occur.
• Even trace amounts of impurities can spell
considerable economic loss, as the entire
production batch then has to be discarded
and the production process has to be
restarted from scratch with the cultivation
of new cells.
5. Formulation and filling
(To bring the protein product into a stable (2y; 2-8°C),
applicable and marketable form)
Biotech products manufacturing
• Because of the sensitive nature of most
biopharmaceuticals, their dosage forms
are limited to injectable solutions.
....ATG Human Gene Sequence STOP...
Cloning into DNA Vector
Transfer into Host Cell Expression
e.g., bacterial or mammalian cell
DNA Vector
ATG
Fermentation
Stop
Downstreaming/ Purification
Different Protein Manufacturers use...
(Probably) a different DNA vector
A different fermentation process
A different downstreaming protocol
Different in-process controls
Maybe the same gene sequence
A different recombinant production cell
Proteins
Proteins Molecular Size and 3-D Structure
M. Clark, http://www-immuno. path. cam.ac.uk/~mrc7/
Antibody (IgG) molecule
Interferon-
Aspirin
Proteins produced by different manufacturers are essentially
different
Source: H. Schellekens (2005) FDA/DIA Scientific Workshop on Follow-on Protein Pharmaceuticals
Biosimilars
Biosimilars
Anything Can Be Reverse Engineered and Copied…
however, some things are much safer than others.
Current Regulatory
Concerns for Biotech Products • Testing
– Endotoxins
– Glycosylation / Glucosylation
– Deamidation
– Aggregates
– Stability
– Product Specs (potency, strength, etc.)
– Mutation
– Mycoplasma
• Validation – DNA
– Genetic Stability
– Host Cell Proteins
– Use of immortalized cell lines
– Intrinsic Virus
– Extrinsic Virus
– Immunogenicity
– Reproducibility of process
– Small molecule removal
• Recent Concerns – Prions
– Leachables / Extractables
Some Issues with Biologics
• Inherent complexity (contributions from uncharacterized minor components)
• Inherent instability (deamidation, proline isomerization) • Immunogenicity and Consequences:
– Limited utility of preclinical animal studies
– Hypersensitivity (systemic / local)
– Enhanced clearance
– Reduced effectiveness (neutralizing)
– Immune complexes
– Inhibition of endogenous protein (e.g., PRCA)
– Limited utility of other (biotech) products
• Unpredictable nature of PK/PD and lack of clinical correlation
Release Tests
Extended Characterization
Process
Adapted from: S. Koszlowski & P. Swann (2006) Adv. Drug Delivery Revs. 58, 707-722
How Much of the Iceberg is Visible?
Advantages in terms of
efficacy and safety
• efficacy and safety.
• Thanks to their structure, proteins have a strong affinity for a specific target molecule.
• dangerous interactions with other drugs as well as side effects are rare..
• Biopharmaceuticals are unable to penetrate into the interior of cells, let alone into the cell nucleus, where many carcinogenic substances exert their dangerous (side) effects.
Mechanisms of Action of
Immunosuppressive Drugs
Structures of
Immunosuppressive Antibodies
Muromonab-CD3 (monoclonal)
Basiliximab (chimeric monoclonal)
Daclizumab (humanized monoclonal)
Antithymocyte globulin (polyclonal)
Mouse
Human
Rabbit, Equine
Mechanisms of Action
T-Cell
Activation
T-Cell
Proliferation
Signal 2: Costimulation Signal 3:
IL-2R
IL-15
Resting
DC
DC
Maturation
Daclizumab
Basiliximab
CsA
Tacrolimus
Muromonab-CD3
MMF
Signal 1: MHC/peptides
Recognition by TCR
MHC TCR
MMF
Steroids
MMF
Sirolimus
T-Cell
Growth
Factors
B7
CD40
CD28
CD40L
Sirolimus
Adapted with permission from Professor Dr. Walter Land and M. Schneeberger, University of Munich, Germany.
Latest Developments
ERYTHROPOESIS and the
Pathophysiology
of Anemia in CKD
Erythropoiesis:
Role of Erythropoietin
Erythropoietin
Erythroblasts
Reticulocyte
BFU-E CFU-E
Apoptosis
without
erythropoietin
BFU-E: Burst-Forming Unit-Erythroid CFU-E: Colony-Forming Unit-Erythroid
Red blood cells
Fisher. Exp Biol Med 2003; 228: 1–14
MOA & Fate of EPO
Stimulation of Erythropoiesis
by Endogenous Epoetin
Stimulation of Erythropoiesis
by Recombinant Epoetin
C.E.R.A.
a continuous erythropoietin receptor
activator
MIRCERA®
• Innovative agent
• MIRCERA® is the
first continuous
erythropoietin
receptor activator
for treatment of
anemia Molecular weight
~60 000 Da
MIRCERA® A continuous erythropoietin receptor
activator
Macdougall et al. ASN 2003 The image represents an artist’s view of a
possible structure for MIRCERA®
MIRCERA® Has Distinct Properties That
Suggest Different Binding to Receptor
MIRCERA® MIRCERA® MIRCERA®
MIRCERA® MIRCERA®
Continuous Stimulation of
Erythropoiesis by MIRCERA®
• Isolated 1989
• Flavivirus
Hepatitis C Virus
Healthy Liver Cirrhosis
PEGylation
• PEG = Polyethylene Glycol polymers
• Inert, non-toxic and water soluble
• PEG attached to IFN to increase
bioavailability
PEGASYS® -Characteristics of 40 KD
Branched PEG-IFN
• High molecular weight
– Low Vd
– Once weekly
– Single dose
• Strong amide bond to IFN
– stable in solution
– Ready to use pre filled syringe
Time
Serum
IFN
Levels
(U/mL)
Optimizing Interferon Kinetics
1 week
“optimised” IFN
2nd Dose
RA is a systemic and
polyarticular disease
Long-term disability
Joint destruction due to loss of cartilage and bone in RA
Cellular changes in the joint
• Pannus formation
– Accumulation of synovial infiltrate
(including CD4+ T cells, macrophages and B
cells)
• Chronic polyarticular effects
– Destruction of cartilage
– Increased bone resorption by osteoclasts
leads to
loss of bone
Firestein GS. Nature 2003;423:356–361
The role of B cells in the
pathophysiology of RA
Autoantibody
production
Cytokine
production
Choy, Panayi. N Engl J Med 2001;344:907–916; Dörner, Burmester. Curr Opin Rheumatol 2003;15:246–252;
Shaw, et al. Ann Rheum Dis 2003; 62 (Suppl. 2):ii55–59; Takemura, et al. J Immunol 2001;167:4710–4718;
Edwards, et al. Immunology 1999;97;188–196
Antigen
presentation
As highly efficient antigen
presenting cells, B cells may
contribute significantly to
T cell responses in RA
Autoreactive B cells produce
autoantibodies that may help
drive the disease process in RA
Activated B cells may
produce cytokines known
to promote inflammation
in RA
The role of B cells in the
pathogenesis of RA
Rituximab selectively targets
CD20-positive B cells Antigen-independent phase Antigen-dependent phase
Activated
B cell
Plasma
cell
Secreted
IgG, IgA,
IgE, or IgM
Mature
B cell
Pro-B cell Pre-B cell Immature
B cell
Surrogate
light chain IgM IgM IgD
IgM, IgD,
IgA, or IgE
Adapted from Sell et al. Immunology, Immunopathology, and Immunity. 6th ed. 2001; Roitt et al. Immunology. 6th ed. 2001;
Tedder et al. J Immunol 1985;135:973.
Stem cell
CD19
CD20
Rituximab (MabThera®/Rituxan®):
The first selective B cell therapy
for RA
Rituximab MOA
Silverman, Weisman. Arthritis Rheum 2003;48:1484–1492; Silverman, Carson. Arthritis Res Ther 2003;
Lund et al. Curr Dir Autoimmun 2005; Duddy et al. J Immunol 2004
Cell-mediated
cytotoxicity Complement-mediated
B cell lysis
Promotion
of apoptosis
2 large green dots
Amplification of red
signals
Coamplification of a
CEP-17-similar gene
fragment
Result:
HER2
Amplified !
Amplified case, Ratio: 6.3
Tumor Cell
Fc
The antibody binds via its Fab portion...
Antibody-Dependent Cellular
Cytotoxicity (ADCC)
…and recruits immune effector
cells via its Fc part
Lysis of target cell
Fab
Introduction to Angiogenesis
Role of Angiogenesis in
Cancer?
Tumours Requires Angiogenesis
• Role in Cancer
• Research has discovered
& demonstrated that one
of the critical events
required is the growth of
a new network of blood
vessels
• Hence the role of
angiogenesis in cancer
Tumours Requires
Angiogenesis
VEGF Receptor
EGF
IGF-1 PDGF
IL-8
bFGF
Hypoxia COX-2
NO Oncogenes
VEGF release Binding and activation of VEGF receptor
H2O2
Proliferation Survival Migration
ANGIOGENESIS Permeability
Increased expression (MMP, tPA, uPA, uPAr,
eNOS, etc.)
– P
– P
P–
P–
IGF = insulin-like growth factor; PDGF = platelet-derived growth factor
Advantages in terms of
efficacy and safety
• efficacy and safety.
• Thanks to their structure, proteins have a strong affinity for a specific target molecule.
• dangerous interactions with other drugs as well as side effects are rare.
• Biopharmaceuticals are unable to penetrate into the interior of cells, let alone into the cell nucleus, where many carcinogenic substances exert their dangerous (side) effects.
Future developments
• MoA + Chemical Molecule
• Production of vaccines using PLANTS
• Future Disease prediction
Thank You !