Overview of Upstream and Downstream Processing of Biopharmaceuticals

Transcription

1 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 1

2 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

3 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

4 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

5 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

6 Design criteria Concentration Productivity (volumetric, specific) Yield/ conversion Quality Purity Sequence Glycosylation Activity (in vitro, in vivo) 6

7 Design criteria for pharmaceutical product Order of importance Quality Concentration Productivity Yield/ Conversion High added value products 7

8 Design criteria for bulk product Order of importance Concentration Productivity Yield/ Conversion Quality Low added value products 8

9 Clear idea of product Selection of producing organism USP Biomass-product separation Product purification DSP Effluent recycle/disposal Strain screening Formulation medium requirements Strain improvement (molecular biology) Concentration, crystallization, drying Fill-Finish Medium optimization Small scale bioreactor Cultures (batch, fed- batch, continuous) Process integration Storage properties, stability Field trials Process control requirements Scale- up (>100 litre) Process kinetics (productivity etc.) Are yields, conversion, productivity ok? DSP FDA approval Product licence Marketting Sales 9

10 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/ posttranslational 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

11 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

12 Type of bioreactor Depends on: Anchorage dependence or suspension adapted, Mixing- homogeneous conditions, absence of nutrient and temperature gradients Mass transfer particularly (OTR = k L a (C * -C L ) Cell density (q O2.x = OUR) CHO and BHK q O2 = pmol/cell/h Shear resistance CIP/SIP Validation issues 12

13 Type of bioreactor Stirred tank reactor (STR) Membrane reactor Fixed-bed reactor Fluidized-bed reactor (FBR) Disposable reactors 13

14 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 10 6 cells/ml. Aim: to achieve high cell density cultures increase overall process productivity PGA, propylene-glycol-alginate 14

15 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

16 Feeding regimes F S 0 F S F S V Continuous V F S 0 Perfusion Batch Fed- batch V F S 16

17 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

18 Process-related related contaminants DSP- the challenge Product-related contaminants 18

19 Dose-Purity relationship Purity hgh 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

20 DSP USP- Culture harvest (product mg/l) Purity Cell separation Capture Volume Intermediate purification Polishing Fill-Finish 20

21 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

22 Chromatography STREAMLINE INdEX CHROMAFLOW BPG FineLINE BioProcess Stainless Steel 22

23 Filtration Reverse Osmosis Nanofiltration Ultrafiltration Microfiltration pore size (microns) Approx. molecular weight (globular protein) Dead end filtration Cross-flow filtration Attention: fouling, membrane polarization, cost, protein aggregation/ precipitation, degradation 23

24 Filtration 24

25 Generic monoclonal antibody production scheme ceramic hydroxyapatite (flow through mode) 25

26 School of Biotechnology Bioprocess Engineering Group Molecular Biology Microbiology PAT On- line monitoring Animal cell Culture Micro- and Nanoencapsulation Integrated bioprocessing Environmental engineering Immunology Bioinformatics, genomics, proteomics etc. Natural and Recombinant products 26

27 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

28 Thank you for your attention Any questions? 28