1. Biopharmaceutical Separation Processes
Chapter 11 Bioprocess Engineering Book
Terry A. Ring
Chemical Engineering
University of Utah

2. Bio Separations are the biggest costs in Biopharmaceutical processing
More so than the reaction to make the biopharmaceutical!

3. Separation Steps – Vary with size and chemical nature of product and environment it is found in
Recovery
Product is at a low concentration
Separation cost is related to initial concentration
Co=100 g/L => $2/kg
Co = g/L => $100,000,000/kg
Purification
Drugs are desired in high purity
Compounds can be
heat sensitive
Osmotic pressure sensitive
pH sensitive
Product of Reaction
Cells & Broth & Nutrients
Lots of water
Desired Products
In biomass
Extracellular component
Intracelular component
Expressed into broth
Location dictates separation methods

4. Separation Technology Range of Applicability

7. Enzyme Separation Separation of insoluble products and other products
Primary isolation or concentration of product
Purification or removal of contaminating chemicals
Product preparation e.g. drying

8. Separation of Cellular Material
Two Cases
Biomass contains Product
Remove the excess liquid
Separated Liquid contains Product
Remove solid waste
Pretreatement
pH adjustment
Ionic strength adjustment
Addition of
Coagulants
Flocculants
Filtration
Vacuum filtration
Rotary vacuum filtration
Micro-filtration
Ultrafiltration
Centrifugation
Continuous and Batch
Coagulation and flocculation

9. Filtration Add filter aid Prevent compressible cake
V= volume of filtrate, A=filter area, rm= resistance of filter medium, C=cake weight per unit volume of filtrate, α= specific resistance of cake, μ=filtrate viscosity

10. Rotary Drum Filter
the drum rotates at a constant speed (n rps) and only a fraction of drum-surface area is immersed in suspension reservoir (φ). The period of time during which filtration is carried out is j/n per revolution of the drum.
V’=filtrate volume per unit time (volume/time) and V’/n represents the volume of filtrate filtered for one revolution of the drum.

11. Centrifugation βo>1 ~1.6 Force Balance on Particle in Fluid
Gravity = Drag + Buoyant R3
Rep<1
Gravity Settling Centrifugal Settling
Dr/dt =[R3-R1]/dt = Uo
t=Vc/QC Uo
ω= angular velocity (rad/s)

12. Disc Geometry

13. Coagulation and Flocculation
Coagulation = add salts or acid/base
Flocculation = add polymer

14. Cell Disruption = release intercellular product
Mechanical Methods
Non-Mechanical Methods - Lysing
Ultrasonic sonicators
Presses
Gaulin-Manton
French
Cell paste
Rannie HP homogenizer
Dyno-mill
Bead mill
X-press
Osmotic Shock
Treatment with solvent
Freeze-Thaw
Ice crystals break cell wall
Freeze drying
Chemical Methods
Enzymes
EDTA (extracts Ca+2 ions)
Followed by ultra centrifugation or ultra filtration

15. Separation of Soluble Products
Liquid-Liquid Extraction
Mixer-Settler
Rejection Ratio
n=No. Stages
Podbielniak

16. Separation of Soluble Products
Dissociation Extraction = extraction of Ionized Species
Requires pH adjustment in one or both liquids

17. Protein
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues.
Most proteins consist of linear polymers built from a series of up to 20 different L-α-amino acids.
Generally 2 Dissociable Groups
N-H
C=O

18. Aqueous 2 phase Extraction for proteins
Protein Partition Coefficient, Kp= exp(A Mwprotein/T)
Phase 1- Polyethylene glycol/H2O
Phase 2 – Dextran/H2O
Mixer/Phase Separation
Phase Separation
Decantation
Centrifugation
Ultra filtration
Add KH2PO4 improves Kp
Add ion-exchange improves Kp
2 aqueous phase affinity partition extraction

19. Protein Precipitation
Salting out
Salt (NH4)2SO4 Addition at high ionic strength
Solubility reduction
Low T < -5°C
Add organic solvents

20. Precipitation of Proteins
Salting Out
Reduce Temperature add Organic Solvent
S= Solubility, So= I=0
Ds = dielectric constant of water- solvent solution

21. Isoelectric Precipitation
When pH = pI, protein becomes free of charge and precipitation takes place
K1 acid dissociation constant for functional group on the protein
K2 acid dissociation constant for functional group on the protein

22. Adsorption – Liquid passing through a packed bed of solid
Equilibrium between liquid and solid phase
Packed Bed Flow Equation
Mass Transport to solid surface
Combined Equation
Solve Numerically for
where CL* and CS* are equilibrium concentrations of the solute in liquid and solid phases, respectively.
A type of Freundlich adsorption isotherm, with KF and n being empirical para-meters and n being greater than 1.

23. Adsorption Column Results
Stripping or Regeneration
Wash Out Column
Remove impurities
Loaded Bed is unloaded
Column is regenerated for another adsorption step
Breakthrough Curve

24. Dialysis Separation of Low Mw solutes Selective Membrade
100<Mw<500 organic acids
10<Mw< 100 ions
Selective Membrade

25. Reverse Osmosis Over come Osmotic Pressure
Πos = (RT/Ṽ1)ln[a1°/a1]~(RT/Ṽ1)n2
Ṽ1= molar volume of the solvent
n2 = mole fraction solute

26. Ultra Filtration/Membranes when Macromolecules are present
Slowly diffusion Macromolecules
Form Gel at membrane surface
Diffusion through the gel is very slow
Cross flow Filtration Prevents Gel

27. Membrane Configurations

28. Chromatography Separation based upon Retention Time in a packed bed
Stationary Phase
Mobile Phase
Types Chromatography
Size Exclusion
(large molecule leave first)
Adsorption
Ion exchange
Liquid-Liquid Partitioning
Gel Filtration
Affinity
Hydrophobic
High Pressure Liquid

29. Affinity Chromatography

30. Separation Resolution
Yield (assuming Gaussian Peak)
ν=superficial Velocity
d= bead diameter
l = column length
D=Taylor Diffusion Coef.

31. Chromatography Flow Equation Movement of solute
M is the amount of adsorbent per unit volume of the column (mg/mL), f(CL) is the adsorption isotherm

32. Electrophoresis
the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
Governing Equations

33. Drying Products