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 10 cells within 24 hours. 21
• 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.
Biotech products manufacturing 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)
• 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.
Biotech products manufacturing 5. Formulation and filling (To bring the protein product into a stable (2y; 2-8째C), applicable and marketable form)
• Because of the sensitive nature of most biopharmaceuticals, their dosage forms are limited to injectable solutions.
Different Protein Manufacturers use... ....ATG Human Gene Sequence STOP...
ATG Cloning into DNA Vector
Stop DNA Vector
Maybe the same gene sequence
(Probably) a different DNA vector Transfer into Host Cell Expression
A different fermentation process
Different in-process controls
Fermentation
A different recombinant production cell e.g., bacterial or mammalian cell
Downstreaming/ Purification
A different downstreaming protocol
Proteins
Proteins
Molecular Size and 3-D Structure Aspirin
Interferon-ď Ą
Antibody (IgG) molecule M. Clark, http://www-immuno. path. cam.ac.uk/~mrc7/
Biosimilars Proteins produced by different manufacturers are essentially different
Source: H. Schellekens (2005) FDA/DIA Scientific Workshop on Follow-on Protein Pharmaceuticals
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
How Much of the Iceberg is Visible? Release Tests Extended Characterization
Process
Adapted from: S. Koszlowski & P. Swann (2006) Adv. Drug Delivery Revs. 58, 707-722
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 Mouse Human Rabbit, Equine
Muromonab-CD3 (monoclonal)
Daclizumab (humanized monoclonal)
Basiliximab (chimeric monoclonal)
Antithymocyte globulin (polyclonal)
Mechanisms of Action Resting DC
MMF Steroids
T-Cell Proliferation DC Maturation
T-Cell Activation MMF
MMF Sirolimus
Signal 2: Costimulation B7
CD28
CD40
CD40L
MHC
TCR
Signal 3: IL-2R IL-15 T-Cell Growth Factors Sirolimus
Signal 1: MHC/peptides Recognition by TCR
CsA Tacrolimus Muromonab-CD3
Daclizumab Basiliximab
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 Reticulocyte
BFU-E
CFU-E
Erythroblasts
without erythropoietin
Apoptosis 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
MIRCERA速 C.E.R.A. a continuous erythropoietin receptor activator
MIRCERA® A continuous erythropoietin receptor activator • Innovative agent • MIRCERA® is the first continuous erythropoietin receptor activator for treatment of anemia Molecular weight ~60 000 Da The image represents an artist’s view of a possible structure for MIRCERA®
Macdougall et al. ASN 2003
MIRCERA® Has Distinct Properties That Suggest Different Binding to Receptor MIRCERA®
MIRCERA®MIRCERA®
MIRCERA®
MIRCERA®
Continuous Stimulation of Erythropoiesis by MIRCERA®
Hepatitis C Virus • Isolated 1989 • Flavivirus
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
Optimizing Interferon Kinetics “optimised” IFN Serum IFN Levels (U/mL)
2nd Dose
Time 1 week
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
The role of B cells in the pathogenesis of RA Antigen presentation
As highly efficient antigen presenting cells, B cells may contribute significantly to T cell responses in RA
Autoantibody production
Autoreactive B cells produce autoantibodies that may help drive the disease process in RA
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
Cytokine production
Activated B cells may produce cytokines known to promote inflammation in RA
Rituximab selectively targets CD20-positive B cells Antigen-independent phase
Surrogate light chain
Stem cell
Pro-B cell
Pre-B cell
IgM
Immature B cell
Antigen-dependent phase
IgM
IgD
Mature B cell
IgM, IgD, IgA, or IgE
Activated B cell
CD19 CD20
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.
Secreted IgG, IgA, IgE, or IgM
Plasma cell
Rituximab (MabThera速/Rituxan速): The first selective B cell therapy for RA
Rituximab MOA Complement-mediated B cell lysis
Cell-mediated cytotoxicity
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
Promotion of apoptosis
Amplified case, Ratio: 6.3 2 large green dots
Amplification of red signals Coamplification of a CEP-17-similar gene fragment
Result:
HER2 Amplified !
Antibody-Dependent Cellular Cytotoxicity (ADCC) Fc The antibody binds via its Fab portion...
Fab
…and recruits immune effector cells via its Fc part
Tumor Cell
Lysis of target cell
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 IGF-1 EGF
PDGF H2 O 2
IL-8 bFGF
Binding and activation of VEGF receptor
VEGF release
Hypoxia COX-2 NO Oncogenes
Increased expression (MMP, tPA, uPA, uPAr, eNOS, etc.)
P–
–P –P
P–
Survival
Proliferation
Migration
ANGIOGENESIS IGF = insulin-like growth factor; PDGF = platelet-derived growth factor
Permeability
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 !