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Transcript
•Large organic compounds which are responsible for catalyzing biochemical reactions, as well as providing structure, mobility, and function for cells. Characterization of Protein-Surface Interactions to Improve Production of Recombinant Proteins Tensiometry •Tensiometry is based upon a simple force balance School of Chemical, Biological, & Environmental Engineering CHE 415-416 Senior Laboratory •Proteins contain different amino acids, each of which contribute to protein structure and function. Regions of the protein are polar and others are hydrophobic; these areas make the protein unique but can cause problems during industrial production of these proteins. F = l cos Warren Gray, Trevor Thompson Problem Statement •Our recombinant protein (produced via genetic engineering) is used to treat a blood disease and is manufactured commercially. However, interfacial interactions upstream in the production process yield significant protein loss, increasing the cost of the drug and reducing availability. Examining adsorption kinetics will enable the control of these interactions, decreasing the cost of production and improving the availability of the drug. •When a protein is in the bulk liquid, interactive regions order the water molecules around it, decreasing the entropy (disorder, “S”) of the system. A system seeks to achieve a minimum of free energy (“G”), defined as: G = H – TS . The protein will move to the interface (adsorb) to increase the entropy, thereby minimizing the free energy. Additionally, there is a Heat of Adsorption (“H”) associated with this process. This also contributes to minimizing free energy. Unfortunately, the foreign environment causes the protein to deform and denature, losing much of its ability to function. When proteins adsorb to the airwater interface, they take the place of water molecules and decrease the measured surface tension at this interface. 1 Region 1: Very low surfactant concentrations · · · Protein Protein Surfactant predominantly in bulk liquid phase Limited surfactant-surface interaction Limited surfactant-protein interaction 2 3 4 Where: F = Measured Force (mN) l = Wetted Perimeter (m) γ = Surface Tension (mN/m) θ = Contact Angle (degrees) •Dynamic tensiometry uses a continuous sampling technique in order to monitor changes in surface tension over time Theoretical Surfactant/Protein Behavior 5 Steady State Surface Tension What are proteins? Region 5: Very high surfactant concentrations · · · · · Surfactant in bulk liquid phase, at interface, and at protein surfaces Appreciable surfactant-surface interaction Appreciable surfactant-protein interaction Protein completely removed from interface Surfactant forms micelles in solution Protein Concentration of Surfactant Region 2: Low surfactant concentrations · · · What is surface tension? Surfactant in bulk liquid phase and at interface Appreciable surfactant-surface interaction Limited surfactant-protein interaction Protein Region 3: Moderate surfactant concentrations · · · Protein Protein Region 4: High surfactant concentrations Surfactant in bulk liquid phase, at interface, and at protein surfaces Appreciable surfactant-surface interaction Appreciable surfactant-protein interaction · Surfactant in bulk liquid phase, at interface, and at protein surfaces · Appreciable surfactant-surface interaction · Appreciable surfactant-protein interaction · Surfactant begins to remove and replace protein at interface Protein Surface tension measured here Experimental Results •Initial surfactant experimentation was focused on determining surfactant adsorption kinetics alone, followed by steady-state studies involving mixed surfactant/protein solutions. Pendant PEO Chains Aqueous/ Liquid Phase •Intermolecular forces between the molecules in the bulk experience equal pull on all sides, resulting in a net force of zero. However, molecules at the surface (or interface) experience a significantly greater “pull” from the molecules below them in the liquid than from the gas molecules above them. The net force results in a “pull” towards the bulk liquid (known as surface tension). Polysorbate (Tween) 80 Structure (PEO) (PPO) (PEO) Hydrophobic PPO Chain TM Pluronic F68 Structure Hydrophobic Surface or Liquid-Air Interface Structural comparison of between Tween 80 and F68 surfactants. Surface tension depression, recorded for various bulk concentrations of surfactant, brought about by surfactant adsorption to the liquid-air interface. What causes this behavior? •The resulting graph of an adsorbing substance will show a predictable decrease to steady state. The various slopes of this graph gives important kinetic protein data. •Tween 80 has weaker interaction with hydrophobic surfaces (less aggressive but aggregates are less stable). •F68 has longer chains, so more easily bound up into aggregates. Information and illustrations courtesy of: The Australian MND DNA Bank <http://www.dnamnd.med.usyd.edu.au/> MRC Clinical Sciences Centre <http://europium.csc.mrc.ac.uk/WebPages/Database/Protein/bloodpaper/figure2.gif> Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. 3rd ed. New York: Worth Publishers, 2000. Class Notes, “Module 1,” Dr. Joe McGuire, Oregon State University, Winter 2008 Paradigm Shift, Inc. <http://www.eyefetch.com/image.aspx?ID=452439> Lot Oriel Group <http://lot-oriel.com/site/site_down/sc_sigma703_uken.pdf> Steady-state surfactant in protein solution. Tween 80 is more surface active than F68 at concentrations over 3 ppm. • Experimentation indicates that Tween 80 is more surface active (both kinetically and at steady-state) than F68 at high surfactant concentrations. • Experiment with Tween 80 in bench or pilot-scale bioreactor systems to possibly replace F68 in industrial processes. 58 57 Surface Tension (mN/m) Recommendations •Have you ever seen one of these insects? The Gerridae family (or Water Strider) takes advantage of surface tension. 59 56 55 54 10 IU/mL 53 52 51 50 49 0 Acknowledgements •The group would like to thank Dr. Joe McGuire and Dr. Phil Harding of Oregon State University for their insight and expertise and our industry sponsor for their continued intellectual and material support. 500 1000 1500 2000 Time (s) Typical dynamic surface tension depression exhibited as protein is allowed to adsorb to the liquid-air interface.