EKC 376 /EKC574 DOWNSTREAM PROCESSING OF BIOCHEMICAL AND PHARMACEUTICAL PRODUCTS -- Introduction--

Bioprocessing Bioprocessing deals with the manufacture of biochemicals, biopharmaceuticals, foods, nutraceuticals, and agrochemicals New biologically derived products have been developed, this includes monoclonal antibodies used for the treatment of cancer and multiple sclerosis, plasmids for gene therapy, cytokines and interleukins. Monoclonal antibodies = an antibody produced by a single clone of cells or cell line and consisting of identical antibody molecules Multiple sclerosis = a chronic disease involving damage to the sheaths of nerve cells in the brain and spinal cord Plasmid = a genetic structure in a cell that can replicate independently of the chromosomes Cytokine = a substance such as interferon and interleukin which is secreted by the immune system and has an effect on other cells

The “Chemical Plants” Bacteria

The “Chemical Plants” Plants

The “Chemical Plants” Yeast

The “Chemical Plants” Fungi

The “Chemical Plants” Transgenic Host

Eukaryotic (animal / plant) Cell

Prokaryotic (bacteria) Cell

Bioseparations Many of these products need to be extensively purified before they can be used for their respective applications. Bioseparations engineering refers to the systematic study of the scientific and engineering principles utilized for the large-scale purification of biological products.

Downstream Processing After successful fermentation or enzyme reactions, desired products must be separated and purified. This final step is commonly known as downstream processing or bioseparation, which can account for up to 60 percent of the total production costs, excluding the cost of the purchased raw materials

Nature of Bioseparation Biological products are present in very low concentrations bioseparation has to be very selective in nature stringent quality requirements for products Biological products are susceptible to denaturation and other forms of degradation Many biological products are thermolabile and hence many bioseparation techniques are usually carried out at sub-ambient temperatures Bioseparation is frequently based on multi-technique separation Thermolabile = readily destroyed or deactivated by heat

Characteristic of Bioseparations Variety of products to be separated: Antibiotics, amino acids, enzymes, organic acid and solvents, vitamins, yeasts, growth factors, nucleotides, dextrans… Broad spectrum of separation method can be used: Physical separations, equilibrium controlled separations, rate controlled separations Mode of operations: steady and unsteady states, batch and continuous, cocurrent and counter current Scale of operation: laboratory, pilot plant, large scale Dextran = a carbohydrate gum formed by the fermentation of sugars, used to make a substitute for blood plasma

RIPP Scheme

RIPP Schemes in Bioseparations Removal of Insoluble's/Recovery: Relatively little product concentration Isolation of products: Relatively nonspecific, remove materials of widely divergent properties compared to the product. Appreciable concentration and product quality increases Purification: Highly specific for the product. Remove impurities of similar chemical functionality and physical properties Polishing: Final sequence

RIPP Schemes & Unit Operations Removal of Insoluble's: Filtration and centrifugation Isolation of products: Adsorption and solvent extraction, membrane Purification: chromatography and precipitation Polishing: crystallization, drying

Example: Processing profile, characteristic of antibiotics Product Step Process Conc. (g/l) Quality (%) Harvest Broth Fermentation 0.1-5 0.1-1.0 Removal of Insoluble's Filtration 1.0-5 0.2-2.0 Isolation Extraction 5-50 1-10 Purification Chromatography 50-200 50-80 Polishing Crystallization 90-100

Example: Purification of reagent grade monoclonal antibody

RIPP Scheme Prior to affinity chromatography the cell culture supernatant needs to be cleaned up by membrane filtration or centrifugation so that cells, cell debris and other particulate matter do not clog-up the affinity column. The nearly purified monoclonal antibody obtained by affinity chromatography is further purified by ion-exchange chromatography and polished by gel-filtration to obtain greater than 98% pure product in the solution form. Supernatant = liquid lying above a solid residue after a process

Citric Acid Manufacturing Fermenter Filter Lime Slurry H(2)SO(4) CaSO(4) Spent Beer Mycelium Evaporator Crystallizer Centrifuge Drier Bulk Storage Carbon

RIPP After the fermentation, the first step is filtration to remove insoluble's Adding lime precipitates the calcium salt of citric acid (isolate the product from soluble impurities) Citrate is then purified by conversion back to the acid and by filtration to remove the calcium sulfate The polishing is achieved by crystallization

Basic of Separation

Basic of Separations Size: filtration, membrane separation, centrifugation Density: centrifugation, sedimentation, floatation Diffusivity: membrane separation Shape: centrifugation, filtration, sédimentation

Basic of Separations (cont.) Polarity: extraction, chromatography, adsorption Solubility: extraction, precipitation, crystallization Electrostatic charge: adsorption, membrane separation, electrophoresis Volatility: e.g. distillation, membrane distillation, pervaporation Electrophoresis = movement of charged particles in a fluid or gel under the influence of an electric field Pervaporation = membrane technical method for the separation of mixtures of liquids by partial vaporization through a non-porous or porous membrane

Basic Principles of Engineering Analysis Material Balance Thermodynamic (Equilibrium) Transport phenomena Process and Product Quality Purity Specific activity Yield

Fundamental Principles Mechanical Physical Thermal Chemical Filtration Centrifugation Membranes Classification Purification Agglomeration Washing Absorption Adsorption Crystallization Precipitation Extraction Drying Distillation Rectification Evaporation Chemi-sorption Chemical reaction