Protein Therapeutics. Novo Nordisk Måløv. Outline

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1 Protein Therapeutics Mats Reslow Novo Nordisk Novo Nordisk Måløv Mats Reslow Protein Structure & Biophysics ProteinTechnology Biopharmaceutical Research Outline Proteins as therapeutics 3 examples on Protein Therapeutics Aspects on formulation of proteins Short introduction of Novo Nordisk Novo Nordisk Research&Development 1

2 Biopharmaceuticals Biopharmaceuticals recombinant therapeutic proteins monoclonal antibodies vaccines ~ 160 Biopharmaceuticals have gained medical approval now represent 25% of new pharmaceuticals Examples of Biopharmaceuticals Pharmaceutical R&D Investment is the highest of Major Industries 2

3 Recombinant Pharmaceutical Markets World wide biotech market for recombinant therapeutics Growth ~40% per annum since 1995 Growth will be accelerated by: Genomics, proteomics, bioinformatics Forecast for the market share of therapeutic antibodies in 2010 ~US$ 24 billion Time Lines and Costs for Pharmaceutical Development Leading Products in the Protein Therapeutic Market (US$mn) 3

4 Outline Proteins as therapeutics 3 examples on Protein Therapeutics Aspects on formulation of proteins Short introduction of Novo Nordisk Novo Nordisk R&D Etanercept Factor VIII Lucentis Rheumatoid arthritis Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disorder that causes the immune system to attack the joints, where it causes inflammation (arthritis) and destruction. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility Rheumatoid arthritis Cytokines (chemical mediators) give rise to inflammation of joint synovium Early and intermediate molecular mediators of inflammation include tumor necrosis factor alpha (TNF-_) 4

5 Etanercept (TNF-± binding protein) Etanercept is a fusion protein of two receptor molecules in solution, soluble TNF receptor, that thereby prolongs the half life or survival time of the circulation receptor, and binds TNF in solution, hence preventing the immune response Etanercept for the Treatment of Human Immunodeficiency Virus Associated Psoriatic Arthritis Before After Etanercept In the United States and the United Kingdom, etanercept is comarketed by Amgen and Wyeth under the trade name Enbrel in two separate formulations, one in powder form, the other as a premixed liquid. 5

6 Hemophilia A- blood clotting disorder Hemophilia A is FVIII deficiency (prevalence 1:5000 only in men) Depending on genetic defect, FVIII deficiency can be more or less severe Conc. Classification Symptoms < 1 % Severe Spontaneous bleedings 1 5 % Moderate Bleedings after minor injuries > 5 % Mild Bleedings only associated with surgery, accidents, etc. Normal haemostasis X TF VIIa Xa TF-Bearing Cell Va II IIa VIII/vWF VIIIa IXa TF IXa VIIa X IX V II VIIIa Xa Va Activated Platelet Va Platelet IIa Hoffman et al. Blood Coagul Fibrinolysis 1998;9(suppl 1):S61. Factor VIII 2351 aa protein Synthesized primarily in the liver Secreted as two-chain molecule Half-life around 12 hr Bound to vwf while in circulation 6

7 Bleedings Bleeding often manifests in target joints Target joints: Joints predisposed to recurrent bleeding Result: Painful, persistent bleedings, leading to progressive, irreversible damage of cartilage and bone Disability Hemophilia treatment Earlier treatment with plasma-derived proteins => e.g. HIV transfections Over 10,000 people living with hemophilia contracted HIV from the tainted blood supply. =>Recombinant Factor VIII 1992 Baxter & Genetics Institute Produced by Chinese hamster ovary cells Refacto Refacto-Developed and produced by Biovitrum, Sweden. Licensed to Wyeth Text IV injections 3-7 days a week 7

8 Age-related macular degeneration (AMD) AMD is the leading cause of blindness in people over 50 years of age. Abnormal blood vessels grow in the back of the eye. These vessels leak blood or fluid that causes blurred or distorted vision. Without treatment, vision loss may be quick and severe. Age-related macular degeneration (AMD) Normal vision The same view with age-related macular degeneration Age related macular degeneration (AMD) is the most common cause of blindness in the industrial countries. In the United States alone there are almost 2 million people that are affected by this disease Age-related macular degeneration (AMD) AMD involves the growth of abnormal new blood vessels into the sub-retinal space and retinal pigment epithelium. These weak vessels leak blood or serum, causing damage to the macula, which is crucial for fine vision. 8

9 VEGF-Vascular Endothelial Growth Factor involved in angiogenesis (the growth of blood vessels from pre-existing vasculature). Ranibizumab Anti-VEGF Ranibizumab monoclonal antibody fragment against VEGF. Ranibizumab was developed by Genentech and is marketed in the United States by Genentech and elsewhere by Novartis under the brand name Lucentis. Lucentis (Novartis Ophthalmics) Dose 0.5 mg/month Ranibizumab (10 mg/ml) Injection volume 50 µl Excipients Trehalose Histidin Polysorbat 20 Intravitreal injection 9

10 Therapeutic monoclonal antibodies approved in the US Outline Proteins as therapeutics 3 examples on Protein Therapeutics Aspects on formulation of proteins Short introduction of Novo Nordisk Novo Nordisk R&D Definitions of Protein Stability Therapeutic proteins The maintenance of a defined structural and functional state under recommended storage conditions. 2-8 C Room temperature Pharmaceutical industry normally aim for 2 year storage 10

11 First objective-liquid formulations Ready-to-use pens Instability of Protein Pharmaceuticals Protein aggregation Deamidation Oxidation Disulfide breakage and formation Hydrolysis Factors affecting protein stability Temperature ph Adsorption Salts Metal ions Shaking shearing Protein concentration 11

12 Physical Instabilities/Aggregation Affected by ph Agitation Interfaces Concentration Excipients/buffers Chemical Instability /Deamidation and iso-asp formation Accelerated at high ph and high temperature Chemical Instability / Methionine Oxidation Affected by oxygen, ph, buffer species etc 12

13 Stability & Formulation of a monoclonal antibody at NovoNordisk Often small amounts available ~0.5-1 mg during screening phase => optimize analytical methods Evolution of antibody technology Initial biophysical characterization mab Denaturation temperature Thermofluor (Tm at different ph) CD temp scan Secondary structure Circular dichroism (CD) Aggregates Dynamic light scattering Size exclusion chromatography 13

14 Thermofluor screen SYPRO orange, fluorescent dye to monitor unfoldning transition of proteins Thermoflour analytical equipment 4-5 µg protein to 90 samples Fluorometric scanning mab Two transition peaks (Fab +Fc) 14

15 3 Citronsyre/Natriumcitrat 100 mm 3.5 Citronsyre/Natriumcitrat 100 mm 4 Natrium Acetat 100 mm 4.5 Natrium Acetat 100 mm 5 Natrium Acetat 100 mm 5.5 Natrium Acetat 100 mm 6 Histidin 100 mm 6.5 Histidin 100 mm 7 Imidazol 100 mm 8 Gly-gly 100 mm 9 Gly-gly 100 mm 10 Gly-gly 100 mm Thermoflour screen Additives 100 mm NaCl 0.25 M Sucrose 0.5 M Sucrose 0.5 % Phenol 0.01% Polysorbate % Polysorbate mm NaCl 10% glycerol Thermofluor screen mab Denaturation temperatures Stability optimum ph 7-8 Screen excipients 15

16 Denaturation temperature,td as measured by Dynamic Light Scattering Denaturation temperature in PBS buffer CD temp scan A denaturation temperature of > 60º C was observed Circular Dichroism spectra Spectra indicates a primarily -sheet structure (minimum at 217 nm), typical of that present in IgG 16

17 HPLC on a BIOSEP3000-SEC column from Phenomenex. Flow: 0.8 ml/min, Mobile phase: PBS-buffer ph 7, 215 and 280 nm, inj volume: 20µl. Dynamic Light Scattering No significant amounts of aggregates was observed by dynamic ligth scattering Size Exclusion Chromatography (SEC) No high molecular weigth species (dimers and polymers) was observed by SEC Outline Proteins as therapeutics 3 examples on Protein Therapeutics Aspects on formulation of proteins Short introduction of Novo Nordisk Novo Nordisk R&D 17

18 A few facts about Novo Nordisk February