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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 !

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