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

2. 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

3. The “Chemical Plants”
Bacteria

4. The “Chemical Plants”
Plants

5. The “Chemical Plants”
Yeast

6. The “Chemical Plants”
Fungi

7. The “Chemical Plants”
Transgenic Host

8. Eukaryotic (animal / plant) Cell

9. Prokaryotic (bacteria) Cell

10. 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.

11. 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

12. 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

13. 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

14. RIPP Scheme

15. 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

16. 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

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

18. Example: Purification of reagent grade monoclonal antibody

19. 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

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

21. 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

22. Basic of Separation

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

24. 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

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

26. 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