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 !