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Overview of Upstream and Downstream Processing of Biopharmaceuticals

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Overview of Upstream and Downstream Processing of Biopharmaceuticals Overview of Upstream and Downstream Processing of Biopharmaceuticals Ian Marison Professor of Bioprocess Engineering and Head of School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland E-mail: [email protected] 1 Outline of presentation • Introduction- what is a bioprocess? • Basis of process design • Upstream processing – Batch, fed -batch, continuous, perfusion • Downstream processing – Philosophy – Chromatography – Examples • Conclusions 2 What is a bioprocess? • Application of natural or genetically manipulated (recombinant) whole cells/ tissues/ organs, or parts thereof, for the production of industrially or medically important products • Examples – Agroalimentaire: food/ beverages – Organic acids and alcohols – Flavours and fragrances – DNA for gene therapy and transient infection – Antibiotics – Proteins (mAbs, tPA, hirudin, Interleukins, Interferons, enzymes etc) – Hormones (insulin, hGH,EPO,FSH etc) 3 Aims of bioprocesses • To apply and optimize natural or artificial biological systems by manipulation of cells and their environment to produce the desired product, of the required quality. • Molecular biology (genetic engineering) is a tool to achieve this • Systems used include: – Viruses – Procaryotes (bacteria, blue- green algae, cyanobateria) – Eucaryotes (yeasts, molds, animal cells, plant cells, whole plants, whole animals, transgenics) 4 Importance of process development ‘ Advances in genetic engineering have, over the past two decades, generated a wealth of novel molecules that have redefined the role of microbes, and other systems, in solving environmental, pharmceutical, industrial and agricultural problems. While some products have entered the marketplace, the difficulties of doing so and of complying with Federal mandates of: safety, purity, potency, efficacy and consistency have shifted the focus from the word genetic to the word engineering . This transition from the laboratory to production- the basis of bioprocess engineering - involves a careful understanding of the conditions most favoured for optimal production, and the duplication of these conditions during scaled- up production ’. 5 Design criteria • Concentration • Productivity (volumetric, specific) • Yield/ conversion • Quality – Purity – Sequence – Glycosylation – Activity ( in vitro , in vivo ) 6 Design criteria for pharmaceutical product Order of importance • Quality • Concentration • Productivity • Yield/ Conversion High added value products 7 Design criteria for bulk product Order of importance • Concentration • Productivity • Yield/ Conversion • Quality Low added value products 8 Clear idea of product Biomass-product USP separation DSP Selection of producing organism Product purification Effluent recycle/disposal Strain improvement Strain screening (molecular biology) Concentration, crystallization, drying Formulation medium requirements Fill-Finish Process Medium optimization integration Storage properties, stability Small scale bioreactor Cultures (batch, Field trials fed- batch, continuous) Process control FDA approval requirements Are yields, Product licence conversion, Scale- up (>100 litre) productivity Marketting ok? Process kinetics Sales (productivity etc.) DSP 9 Choice of production cell line- microbes • Bacterial cells – genetic ease (single molecule DNA, sequenced) – high productivity, high µ – Resistance to shear, osmotic pressure, immortal – Negatives: poor secretors, little glycosylation/ post - translational modifications • Yeast – High µ, high cell concentrations, high productivity, good secretors, post-translational modifications, glyco-engineered strains available – Non-mammalian glycosylation, post-translational modifications, complexity of genetic manipulation 10 Choice of production cell line- mammalian cells • CHO/ BHK/HEK/COS…… cells – Advantages • Produce ‘human-like’ proteins • Secrete • Correctly constructed and biologically very active – Disadvantages • Slow growth rate ( µ) • Low cell densities • Low productivity • Shear sensitive, osmotic pressure sensitive, substrate/ product toxicity, apoptosis, cell age Choice of cell line profoundly affects selection of bioreactor, DSP, feeding regime, scale of production 11 Type of bioreactor Depends on: • Anchorage dependence or suspension adapted, • Mixing- homogeneous conditions, absence of nutrient and temperature gradients * • Mass transfer particularly (OTR = k La (C -CL) • Cell density (q O2 .x = OUR) – CHO and BHK q O2 = 0.28-0.32 pmol/cell/h • Shear resistance • CIP/SIP • Validation issues 12 Type of bioreactor Stirred tank reactor Membrane reactor (STR) Fixed-bed reactor Fluidized-bed reactor (FBR) Disposable reactors 13 Animal cell encapsulation CHO cells secreting human secretory component (hSC) 0 days 3 days 12 days Microscope photographs during the repetitive fed-batch culture. Capsules produced with 1.2% alginate, 1.8% PGA, 4% BSA, 1% PEG, initial cell density 106 cells/ml. Aim: to achieve high cell density cultures increase overall process productivity PGA, propylene-glycol-alginate 14 Type of substrate feeding • Depends on anchorage dependence or suspension adapted • OTR (poor oxygen solubility; 5-7 mg/L 25 C) • Cell density (q O2 .x = OUR) • Shear resistance • Stability of product • Productivity • Product concentration • Formation of toxic products • Osmotic stress • Substrate inhibition/ catabolite repression/ diauxic growth • Availability/ Need of PAT (quality by design, consistency) 15 F S 0 F S Feeding regimes F S Continuous V V F S 0 Perfusion Batch Fed- batch V F S 16 Questions • Which regime provides for highest product concentration (titre)? – Which regime provides for highest productivity? • Which regime is used for situations where product is unstable? – Which regime is used when substrates are inhibitory, repressive, mass transfer is limiting? • Which regime is used to design the smallest installation? – Which regime is the easiest to validate? • Which USP is easiest to integrate with DSP? – etc (think up some of your own questions!!) 17 DSP- the challenge Process- related related contaminants Product-related contaminants 18 Dose-Purity relationship Purity 99.997 hGH 99.99 SOD 99.9 EPO 99 Vaccine 95 Diagnostic In vitro 100 mg 1 g 3 g >10 g Lifetime doseage Required Purity as a Function of Dosage 19 USP- Culture harvest DSP (product 10-1000mg/l) Cell separation Purity Volume Capture Intermediate purification Polishing Fill-Finish 20 Purification techniques • Filtration • Precipitation • Liquid-liquid two-phase separation • Chromatography – Size exclusion (gel filtration) – Ion-exchange – Hydrophobic interaction – Reverse- Phase – Hydroxyapatite – Affinity (protein A,G etc, dyes, metal chelates, lectins etc…) – Fusion proteins (tagging, Fc, Intein, streptavidin etc…) 21 Chromatography STREAMLINE™ CHROMAFLOW™ INdEX™ BioProcess™ Stainless Steel BPG™ FineLINE™ 22 Filtration Reverse Osmosis Nanofiltration Ultrafiltration Microfiltration 0.001 0.01 0.1 1.0 pore size (microns) 3 5 10 10 10 7 Approx. molecular weight (globular protein) Dead end filtration Cross-flow filtration Attention: fouling, membrane polarization, cost, protein aggregation/ precipitation, degradation 23 Filtration 24 Generic monoclonal antibody production scheme ceramic hydroxyapatite (flow through mode) 25 School of Biotechnology Bioprocess Engineering Group Molecular Microbiology Biology On- line PAT monitoring Animal cell Culture Micro- and Integrated Nano- bioprocessing encapsulation Environmental engineering Immunology Natural and Bioinformatics, Recombinant genomics, products proteomics etc. 26 Conclusions • Bioprocesses are, or should be, integrated processes designed taking all parts into account to provide the quantity and quality of product required using the least number of steps, in most cost-effective manner. • Holistic approach to process design • Quality by design 27 Thank you for your attention Any questions…………? 28.
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