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Protein drug development Patrik Strömberg

Sobi is an international specialty healthcare company

700 employees in 24 countries

5 core products and 35 partner products

384 M USD turnover 2015

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Dedicated to rare diseases

We are dedicated to rare diseases Our key therapeutic areas are inflammation, genetic diseases and haemophilia We provide access to 35 speciality care products from 24 partners in a pan-European setting

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Protein drug Vaccine

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Disposition • Introduction: proteins and protein drugs – Monoclonal antibodies

• Classification of protein drugs • Important aspects and opportunities for protein drugs • Example 1. EPO • Example 2. TNF blockers

Formula

C33H35FN2O5

Mol. mass

559 g/mol

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Formula

C267H408N72O77S6

Mol. mass

6 063 g/mol

Proteins and protein drugs •

Large macromolecules (>5 kDa) consisting of 20 different L-amino acids and sugar groups (and other modifications) – 10 – 1000 x traditional small molecule drugs

• Complex structures enabling complex functions – Protein-Protein interactions, enzymes, functional domains

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Differences vs. small molecules (N.B. there are exceptions) – Administration by injection (degradation in the GI tract, not absorbed) – Limited distribution (not intracellular)

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Focus: diseases with great medical need, high price level, shorter development timelines and lower attrition (sometimes) 1923 Insulin purified from pigs and cows is introduced (Eli Lilly) 1982 Humant recombinant Insulin produced in E.coli (Genentech /Eli Lilly)

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Formula

C815H1317N233O241S5

Mol. mass

18 396 g/mol *

* Incl. Glycosylation = 37 kDa

Formula

C2532H3854N672O711S16

Mol. mass

55597.4 g/mol (unglycosylated) Enzymes are proteins that catalyze chemical reactions (i.e. increase the reaction velocity). Almost all biological processes within the cell need enzymes to proceed at appreciable rates.

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Biotechnological production • Most protein drugs are produced using recombinant DNA technology – Cloning of gene and/or cDNA – DNA is introduced into host cells (cell line) – Heterologous protein expression

• Frequently used expression systems: – Microorganisms- bacteria (eg. E.coli) and yeast – Eukaryotic cells- insect cells, mammalian cells (eg. CHO) – Transgenic animals and plants

• Protein/process characterization – Complex structures, patterns of posttranslational modifications – Product- and/or process-specific impurities – Important to be able to show: • Identity, Purity, Stability and Consistency of manufacture

Formula

C2224H3475N621O698S36

Mol. mass

51 235 g/mol* * Dimer + glycosylation = 150 kDa

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Gene technology and protein engineering • • • •

”Cut-and-paste” DNA Design and modification of proteins Fusions and chimeric molecules Production by biotechnological processes DNA (chromosome)

Formula

C6416H9874N1688O1987S44

Mol. mass

143 860 g/mol

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Monoclonal antibodies (mAbs) •

Antibodies as drugs – Targeting – variable regions – Stability and effector functions – Fc region – mAbs are usually produced by immunizations in mice

Mouse antibody

• Immunogenicity Chimeric

Humanized (CDR graft)

Fab Fully human

Arthritis Research & Therapy, 2009, 11, 225

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Buss et al. Current Opinion in Pharmacology 2012, 12:615–622

Parker C, Lancet, 2009

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Classification of protein drugs • Substitution therapy: – replacement of defective/absent protein – augmentation of signaling pathway • Insulin, growth hormone, coagulation factors, enzyme replacement • Haematopoiesis (Epo), Stimulation of white blood cells (G-CSF), fertility hormone treatment (FSH), immune regulation (interferon)

• Targeting – Antibodies or related molecules/scaffolds – Natural receptors or regulatory molecules

Rituximab Lantus (insulin) Aranesp (epo)

Enbrel Cerezyme

Aggarwal, Nature Biotechnology, 2014 32 33-39

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La Merie Publishing 2012

Important properties, considerations, and opportunities for a protein drug •

Manufacturing costs (Cost-of-goods, COGS) – Expression systems – Expressions levels – Process (upstream, downstream, analysis, formulation)

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Immunogenicity – Humanization, glycosylation, host cell – Anti-drug antibodies, allergic reactions (anaphylaxis)

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Dosage and pharmacokinetics – – – –

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Route of administration: intravenous (iv), intramuscular (im), subcutaneous (sc) Pharmacokinetics: Plasma half-life, bioavailability Dose regimen “Convenience”

Protein engineering and/or chemical modification Intellectual property rights – Freedom-to-operate and product protection (patents etc.)

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Example 1. Erythropoietin (Epo) • Glycoprotein hormon (growth factor) that regulates the production of erythrocytes (red blood cells) • Naturally secreted from the kidneys • Recombinant Epo has been in use for more than 20 years – Improved novel Epo-variants now available – Epo-doping

• Epo is used to treat anemia as a result of renal failure (or e.g. cancer treatment)

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• 1989: the first Epo drug was approved – Epoeitin alfa (Epogen/Prokrit) – Recombinant protein based on the human Epo sequence • 165 aa (3 glycosylations), 30 kDa

– Produced in CHO cells (hamster origin) – Short plasma half-life (~ 8 h) • Three doses / week

Use of Epo has been associated with Pure Red Cell Aplasia (PRCA) – a rare type of severe anemia. A result of Anti-Epo antibodies (immunogenicity) Elliott S et al. Nature Biotechnology 2003

• 2001: a new Epo drug with improved pharmacokinetics was launched – Darbepoetin (Aranesp) – Hyperglycosylated • 5 glykosylations, 37 kDa

– Prolonged half-life (25 h) • One dose / week

Elliott S et al. Nature Biotechnology 2003

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• 2001: a new Epo drug with improved pharmacokinetics was launched – Darbepoetin (Aranesp) – Hyperglycosylated • 5 glykosylations, 37 kDa

– Prolonged half-life (25 h) • One dose / week

Macdougall IC et al J Am Soc Nephrol 1999 Egrie & Browne Nephrol Dial transplant 2001

Strategies to extend the plasma half-life of recombinant proteins

Kontermann R.E., (2011) Current Opinion in Biotechnology 22: 868–876

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• 2002: Shire tried to launch an Epo product produced in human cells (not hamster) – Epoeitin delta (Dynepo) • Human glycosylation pattern, less immunogenic?

– Still short half-life, three doses / week • Not marketed anymore

• 2007: PEGylerated Epo with much longer half-life is launched – PEG-epoetin beta (Mircera) – Total mol. weight 60 kDa

MIRCERA (Methoxy polyethylene glycol-epoetin beta)

• 30 kDa PEG conjugated to Lysine

– One dose / month (T1/2 130 h) – Patent disputes with Amgen • Not sold in the US

http://www.roche.com/products/product-details.htm?type=product&id=83

PEGylation

Veronese FM 2005

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• 2012: approval of a product that addressed both potential problems with pharmacokinetics and immunogenicity – Peginesatide (Omontys® Affymax) – Synthetic, pegylated dimeric peptide (not biotech ) • 5 kDa peptide plus 40 kDa PEG

– Peginesatide binds the erythropoietin receptor but does not share the Epo sequence • No risk of cross-reactive immunogenicity (and PRCA)

– Half-life ~50 h, 1 dose / month

• But in February 2013 Omontys was recalled – Allergic reactions (hypersensitivity), sometimes anaphylactic chock, in 0.2 % of patients

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Example 2. TNF blockers • Tumour necrosis factor (TNF) is a proinflammatory protein (cytokine) that signals by binding to TNF receptors • Involved in many inflammatory and autoimmune diseases – Reumatoid Arthritis (RA): chronic inflammation in joints

• Anti-TNF treatment with protein drugs since 1998 – Antibodies and antibody derived/related molecules

Monoclonal antibodies • Infliximab (Remicade) 1998 – Chimeric IgG monoclonal antibody • Humana constant (70%) and mouse variable regions (30%)

– Dose: 3 mg/kg iv infusion every 6-8 week

• Adalimumab (Humira) 2002 – Fully human IgG monoclonal antibody • Generated with in vitro technology (phage display)

– Dose: 40 mg sc / 2 weeks

• Golimumab (Simponi) 2009 – Fully human IgG monoclonal antibody • Generated in transgenic mice (HuMAb-Mouse)

– Dose: 50 mg sc / 1 month

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https://www.simponihcp.com/rheumatoid-arthritis/dosing

Fusion proteins and antibody fragments • Etanercept (Enbrel) 1998 – Fully human fusion protein • Extracellular part of the TNF receptor and the Fc part of human IgG

– Dose: 50 mg sc / 1 week

• Certolizumab pegol (Cimzia) 2008 – Humanized antibody Fab' fragment, conjugated with 40kDa PEG • Fab' fragment produced in E. coli bacteria followed by purification and PEG conjugation

– Dose: 400 mg sc / 4 weeks

http://www.cimziahcp.com/how-to-use-prefilled-syringe

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D. Tracey et al. / Pharmacology & Therapeutics 117 (2008) 244–279

Summary •

Manufacturing costs

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Immunogenicity

– E.coli production (Certolizumab pegol) – Fully human mAb (Adalimumab) – Production in human cells (Epoetin delta) – Allergic reactions (Peginesatide)

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Dosage and pharmacokinetics – Epo – normal dosage 3 injections / week •

Hyperglycosylated: 1 dose /week; PEGylated: 1 dose /month

– Anti-TNF •

Golimumab: subcutaneous injection, 1 dose / month

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Intellectual property rights

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Convenience

– Mircera (PEGylated Epo) infringed on Amgen patents – A subcutaneous injection that can be self-administered by the patient (e.g. on a monthly basis) using a pre-filled syringe, designed to fit a reumatic person, and that can be stored at room temperture

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