Future trends and perspectives in modern pharmaceutical biotechnology

FUTURE TRENDS AND PERSPECTIVES IN MODERN PHARMACEUTICAL BIOTECHNOLOGY Prof. Borut Štruk elj Faculty of Pharmacy, UL, Slovenia BWP, EMEA London Ph. Eur. Strasbourg

Pharmaceutical biotechnology Traditional biotechnology -secondary metabolites -antibiotics -steroids -vitamins, etc Modern biotechnology -recombinant proteins -monoclonal antibodies -gene therapy -transgenic organisms

What is a modern pharmaceutical biotechnology? keyword: “ PHARMACEUTICAL BIOTECHNOLOGY” GOOGLE 1996: app. 35.000 hits GOOGLE 2006: app: 3.220.000 hits ! Pharmaceutical biotechnology: the use of living things or parts in order to create or modify drugs Modern pharmaceutical biotechnology: the use of living things or parts by means of recombinant DNA technology in order to create or modify drugs and physiological targets

From the starting point of protein pharmaceuticals…. 100 Years ago Serum Therapy 1893 Emil von Behring immunizing a “ serum-horse”

… .to modern biotech

Trends and perspectives

1 st generation of biopharmaceuticals

2 nd generation of biopharmaceuticals

Introduction of biosimilars

Novel technologies for R&D and advanced drug delivery

Gene therapy and tissue engineering

Personalised drug therapy

Biopharmaceuticals or protein drugs have proven to be safe and effective therapies in many disease indications The 1 st generation biopharmaceuticals Replacement therapies (mimic the normal function of a protein) “ Antagonist” therapies (in which protein function is inhibited - usually antibodies) Many successful products: Erythropoietin, insulin, human growth hormon (HGH), interferons (IFNs), granulocyte colony-stimulating factor (G-CSF), etc.

Cytokines Hormones Coagulation factors/ Antibodies Vaccines and antagonists and peptides Inhibitors Abciximab Hepatitis Vaccine Interferon alfacon-1 Insulin Eptacog alfa Adalimumab LymeDiseaseVaccine Interferon alfa-2a Antihemophilic Alemtuzumab Diphtheria/Tetanus/ Interferon alpha-2b Factor (3) Arcitumomab Pertussis Vaccine Moroctocog alfa Basiliximab Rotavirus Vaccine (FVIII mutein) Bevacizumab Interferon beta-1a Epoetin alfa Nonacog alpha Gemtuzumab Enzymes Interferon beta-1b Epoetin beta Desirudin Ibritumomab Alteplase (t-PA) Interferon gamma-1b Lepirudin Infliximab Reteplase Aldesleukin (IL-2) Drotrecogin-alfa Palivizumab Tenecteplase (TNK-t-P) Filgrastim (G-CSF) Follitropin alfa (Protein C act.) Rituximab Monteplase Follitropin beta  1-Proteinase Trastuzumab Dornase-alfa (RNase) Lenograstim (G-CSF) Somatropin i nhibitor Omalizumab Imiglucerase Molgramostim (GM-CSF) Glucagon Efalizumab Agalsidase alfa and beta Sargramostim (GM-CSF) Teriparatide (PTH 1-34) Catuximab Laronidase Tasonermin (TNF-a) Salmon Calcitonin Daclizumab Rasburicase Becaplermin (PDGF-BB) Thyrotropin-alfa Panitumumab Oprevelkin (IL-11) Choriogondotropin A2 Ranibizumab Anakinra (IL-1-RA) Osteogenic Protein-1 Fusion Proteins Dibotermin alpha (BMP-2) Denileukin diftitox Pegvisomant (hGH antagonist) Etanercept Nesiritide (natriuretic peptide) Alefacept Lutropin-alfa 1st generation of biopharmaceuicals

Limitations of the 1 st generation biopharmaceuticals:

Marketed drugs are differently produced nature proteins, with unoptimized physico-chemical properties. Native proteins have not evolved to be human drugs, but to serve a specific function in the body. As such they are often deficient in important drug qualities such as solubility, stability, half-life, potency, recombinant expression yields, reduced immunogenicity.

Drug activity is largely restricted to nature’s existing repertoire of biological activities. Agonists are not easily transformed into antagonists.

The Problem with Proteins

Generally

Very large and unstable molecules (prone to denaturation, a ggregation , p recipitation , a dsorption , d eamidation , o xidation , d isulfide exchange , p roteolysis ...)

Structure is held together by weak noncovalent forces

Easily destroyed by relatively mild storage conditions

Easily destroyed/eliminated by the body

Hard to obtain in large quantities

In the body

Elimination by B and T cells

Proteolysis by endo/exo peptidases

Small proteins (<30 kD) filtered out by the kidneys very quickly

Unwanted allergic reactions may develop (even toxicity)

Loss due to insolubility/adsorption

Desired Drug Properties

Increased physico-chemical stability (thermal stability, longer shelf-life)

Improved solubility and formulation

Improved potency

Reduced immunogenicity

I ncreased proteolytic resistance

Improved half-life in serum

R educed toxicity

Increased bioavailability

Faster or slower onset of action

Elimination of degradation products

Introduction of novel functions

Alternative, non-parenteral routes of delivery (e.g. pulmonary, transdermal, nasal, oral..)

Economically favorable production systems

2nd Generation Biopharmaceuticals: Transforming Proteins into Protein Drugs with Improved Properties Post-production modification derivatization, conjugation (PEGylation , polysialylation, HESylation, fatty acid group derivatization) Amino acid engineering From single mutations to large scale modifications (chimeric or humanized mAbs), introduction/removal of glycosylation sites, introduction/removal of cysteines, introduction of unnatural amino acids, alteration of protease sensitive regions, removal of agretopes to reduce immunogenicity, removal of deamidation-prone Asn, substitution of expoased non-polar amino acids, etc. Protein-protein fusions (IgG, IgG1 Fc, albumin, transferrin, Hsp 65, antibodies or their fragments) New drug delivery systems (liposomes, nanoparticles, microparticles)

PEGylation is currently the main approach for improving protein therapeutic value fulfilling many of the required protein drug properties:

Increases in vivo half life (4-400X)

Decreases immunogenicity

Increases protease resistance

Increases solubility & stability

Reduces depot loss at injection sites

PEGylation In solution the PEG begins to move rapidly, sweeps out a large area (hydrodynamic volume) and protects in this way the protein from environmental influences . Protein  PEG-Protein In vitro activity: 100 % reduced In vivo activity: 100 % enhanced 30–50 nm 5 nm

Future trends : app 500 biotech drugs in clinical trials in 2007 480 are protein-based, 11 gene therapy, 14 antisense DNA or RNAi-based products

2 nd generation: Insulins

2nd generation: Darbepoietin

Proteins vs SCE: complexity Size Structure Modification Stability Epoetin Aspirin Denaturation, Aggregation, Degradation, Oxidation, ... Glycosylation, Acylation, etc.

IgG Antibody ~ 25,000 atoms Aspirin 21 atoms hGH ~ 3000 atoms Large Biologic Small Molecule Drug Large Molecule Drug Car ~ 3000 lbs F16 Jet ~ 25,000 lbs (without fuel) Bike ~ 20 lbs Proteins: Size, Structure & Complexity Complexity Size Source: Genentech

Manufacturing of recombinant Proteins is a complex p rocess DNA Vector Cloning into DNA Vector Large-scale Fermentation Downstreaming Transfer into Host Cell, Expression, Screening/Selection e.g., bacterial or mammalian cell Formulation

Biosimilars Manufacturers: Different Process – different Product Maybe the same genetic sequence (Probably) a different DNA vector A different recombinant cell expression system A different fermentation process A different downstreaming protocol Different in-process controls Maybe a different formulation A second manufacturer uses... DNA Vector Cloning into DNA Vector Large-Scale Fermentation Downstreaming Formulation Transfer into Host Cell, Expression e.g., bacterial or mammalian cell

Two complex biopharmaceuticals cannot be „the same “ but „similar“