Transcript Slide 1

•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.
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Region 1: Very low surfactant concentrations
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Protein
Protein
Surfactant predominantly in bulk liquid phase
Limited surfactant-surface interaction
Limited surfactant-protein interaction
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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
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Steady State Surface
Tension
What are proteins?
Region 5: Very high surfactant concentrations
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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
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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
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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.
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Surface Tension (mN/m)
Recommendations
•Have you ever seen one of these insects? The Gerridae
family (or Water Strider) takes advantage of surface tension.
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10 IU/mL
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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.
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Time (s)
Typical dynamic surface tension depression
exhibited as protein is allowed to adsorb to the
liquid-air interface.