Overview of Upstream and Downstream Processing …

1 Overview of Upstream and Downstream Processing of Biopharmaceuticals1 Ian MarisonProfessor of Bioprocess Engineering and Head of School of Biotechnology,Dublin City University, Glasnevin, Dublin 9, IrelandE-mail: of presentation Introduction- what is a bioprocess? Basis of process design Upstream Processing Batch, fed-batch, continuous, perfusion2 Batch, fed-batch, continuous, perfusion Downstream Processing Philosophy Chromatography Examples ConclusionsWhat is a bioprocess? Application of naturalor genetically manipulated(recombinant) whole cells/ tissues/ organs, or partsthereof, for the production of industrially or medically important products Examples3 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)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.

2 Molecular biology (genetic engineering) is a toolto achieve this4 Systems used include: Viruses Procaryotes (bacteria, blue- green algae, cyanobateria) Eucaryotes (yeasts, molds, animal cells, plant cells, whole plants, whole animals, transgenics)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 solvingenvironmental,pharmceutical,indus trial and some products have entered the marketplace, the difficulties of doing so and of complying with Federal mandates of:safety, purity, potency, efficacy andconsistencyhave shifted the focus from the word geneticto 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.

3 Design criteria Concentration Productivity (volumetric, specific) Yield/ conversion6 Yield/ conversion Quality Purity Sequence Glycosylation Activity (in vitro, in vivo) Design criteria for pharmaceutical productOrder of importance Quality Concentration7 Concentration Productivity Yield/ ConversionHigh added value productsDesign criteria for bulk productOrder of importance Concentration Productivity8 Productivity Yield/ Conversion QualityLow added value productsBiomass-productseparationProduct purificationStorage properties,stabilityEffluent recycle/disposalConcentration,crystalliz ation, drying Fill-Finish DSPC lear idea of productSelection of producingorganismStrain screeningFormulation mediumrequirementsMedium optimizationStrain improvement(molecular biology)USPP rocessintegration9 Field trialsstabilityFDA approvalProduct licenceMarkettingSalesSmall scale bioreactorCultures (batch,fed- batch, continuous)Process controlrequirementsScale- up (>100 litre)Process kinetics(productivity etc.)

4 Are yields,conversion,productivityok?DSPinte grationChoice of production cell line- microbes Bacterial cells genetic ease (single molecule DNA, sequenced) high productivity, high Resistance to shear, osmotic pressure, immortal Negatives: poor secretors, little glycosylation/ post-10 Negatives: poor secretors, little glycosylation/ post-translational modifications Yeast High , high cell concentrations, high productivity, good secretors, post-translational modifications, glyco-engineered strains available Non-mammalian glycosylation, post-translational modifications, complexity of genetic manipulationChoice of production cell line- mammalian cells CHO/ BHK/ cells Advantages Produce human-like proteins Secrete Correctly constructed and biologically very active11 Disadvantages Slow growth rate ( ) Low cell densities Low productivity Shear sensitive, osmotic pressure sensitive, substrate/ product toxicity, apoptosis, cell ageChoice of cell line profoundly affects selection of bioreactor, DSP, feeding regime, scale of production Type of bioreactorDepends on.

5 Anchorage dependence or suspension adapted, Mixing- homogeneous conditions, absence of nutrient and temperature gradients12temperature gradients Mass transfer particularly (OTR = kLa (C*-CL) Cell density ( = OUR) CHO and BHK qO2 = pmol/cell/h Shear resistance CIP/SIP Validation issuesType of bioreactorStirred tank reactorMembrane reactor13 Stirred tank reactor(STR)Fluidized-bed reactor(FBR)Membrane reactorDisposable reactorsFixed-bed reactorAnimal cell encapsulationCHO cells secreting human secretory component (hSC)14 PGA, propylene-glycol-alginateMicroscope photographs during the repetitive fed-batch culture. Capsules produced alginate, PGA, 4% BSA, 1% PEG, initial cell density 106 days3 days12 daysAim:to achieve high cell density culturesincrease overall process productivityType of substrate feeding Depends on anchorage dependence or suspension adapted OTR (poor oxygen solubility; 5-7 mg/L 25 C) Cell density ( = OUR) Shear resistance Stability of product15 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)Feeding regimesF SF S0F SVContinuous16 VBatchFed- batchF S0F SVPerfusionQuestions Which regime provides for highest product concentration (titre)?)

6 Which regime provides for highest productivity? Which regime is used for situations where product is unstable? Which regime is used when substrates are inhibitory, 17 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!!)DSP- the challengeProcess-related contaminants18related contaminantsProduct-related contaminantsDose-Purity vitro100 mg1 g3 g>10 gVaccineEPOL ifetime doseageRequired Purity as a Function of DosageDSPCell separationCaptureVolumePurityUSP- Culture harvest(product 10-1000mg/l)20 IntermediatepurificationPolishingFill-Fi nishPurification techniques Filtration Precipitation Liquid-liquid two-phase separation Chromatography21 Chromatography Size exclusion (gel filtration) Ion-exchange Hydrophobic interaction Reverse- Phase Hydroxyapatite Affinity (protein A,G etc, dyes, metal chelates, lectins ) Fusion proteins (tagging, Fc, Intein, streptavidin )ChromatographySTREAMLINE INdEX CHROMAFLOW 22 INdEX BPG FineLINE BioProcess Stainless SteelFiltrationUltrafiltrationMicrofiltr ationReverse size (microns)103107105 Approx.

7 Molecular weight (globular protein)Dead end filtrationCross-flow filtrationAttention: fouling, membrane polarization, cost, protein aggregation/ precipitation, degradationFiltration24 Generic monoclonal antibody production scheme25ceramichydroxyapatite(flow through mode)School of BiotechnologyBioprocess Engineering GroupIntegrated On- linemonitoringMolecularBiologyMicrobiolo gyAnimal cellCulturePAT26 Integrated bioprocessingEnvironmentalengineeringNat ural andRecombinantproductsMicro- and Nano-encapsulationImmunologyBioinformati cs,genomics, Bioprocesses are, or should be, integrated processes designed taking all parts into account to provide the quantity and quality of product 27to 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 designThank you for your attention28 Any