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

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

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 =0.00001 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

Separation Technology Range of Applicability

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

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

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

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.

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)

Disc Geometry

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

Membrane Configurations

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

Affinity Chromatography

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

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

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

Drying Products