An Investigation of the Stabilizing Effects of Surfactants on Biotherapeutics Marianna Fleischman SURF Program 2013 University of Delaware Advisors: Drs. Ron Jones, Tatiana Perevozchikova, and Katie Weigandt
An Investigation of the Stabilizing Effects of Surfactants on Biotherapeutics
Marianna Fleischman SURF Program 2013
University of Delaware
Advisors: Drs. Ron Jones, Tatiana Perevozchikova, and Katie Weigandt
The objective of this research is to find out more about why/how non-ionic surfactants reduce aggregation.
Our Research
• Protein adsorption to interfaces
• Interactions between proteins in bulk solution
Aggregation Particulate/ sediment formation
Immunogenic response triggered
(inflammation)
Toxicity
Air-water Interfaces Langmuir Trough and X-Ray Reflectometry Micro-Flow Imaging
Bulk material Small-Angle Neutron Scattering (SANS)
Potential locations for aggregation:
Our Research
Solid-water Interfaces Micro-Flow Imaging Fluorescence Spectroscopy
Background – Monoclonal Antibodies
• Target specific antigens • Can be engineered in a lab • Effective against autoimmune
diseases and cancer
• Specificity reduces side effects
TRES3D Medical and Scientific Animation
PresenterPresentation NotesAutoimmune disease
they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells
What are surfactants?
Amphiphilic molecules:
Hydrophilic head group
Hydrophobic tail group
Results in unique behavior:
Forming monolayers at interfaces Micellular formation
*At or above the critical micelle concentration (CMC)
How might surfactants help?
Competitive binding at interfaces Encapsulation in micelles
Preferential binding leading to steric hindrance of aggregation
• 4 Surfactants – Triton X-100 – Tween 80 – Octyl-beta-D-glucopyranoside (OG) – Cetyltrimethylammonium Bromide (CTAB)*
• Protein – Immunoglobulin G (IgG)
=4 samples; collected data at multiple concentrations of surfactant (below, at, above CMC)
Bulk Solution Investigation: 10m SANS
Bulk Solution Investigation: 10m SANS
SANS Data: IgG and Two Surfactants
• Dampening of upturn at low q • Some change of solution structure at medium-high q
PresenterPresentation NotesSuppression of higher-order structures
Changes at high q could be due to:competitive scattering of micelles and proteinsselective binding of surfactant to proteinperhaps encapsulation
Look more into: scan of surfactant itselfcontrast-match out one of two species: preferably the surfactant so we can see how structure of protein itself is changing
[INTRO REFLECTOMETRY]
Air/Water Interface Investigation: Xray Reflectometry
0 1 2 3 410-10
10-8
10-6
10-4
10-2
100
Surface RoughnessDensity
Thickness
Refle
ctivi
tyAngle of Incidence (degrees)
Air/Water Interface Investigation: Xray Reflectometry
Protein Adsorption (one of the possible configurations) at air-water interface
LAir
LHead Groups LSolvent
LProtein
LBulk Solvent
LTails
Langmuir Trough Work:
Air/Water Interface Investigation: Xray Reflectometry
1 mg/ml IgG
+Triton X-100 (below CMC)
+Triton X-100 (above CMC)
+IgG (5mg/ml)
Dilate/Compress Barrier
???
Competitive Adsorption
Air/Water Interface Investigation: Xray Reflectometry
Monolayer Moveable barrier
Subphase
Trough
Micro-Flow Imaging
X-Ray Reflectometry
Air/Water Interface Investigation: Xray Reflectometry
Monolayer Moveable barrier
Subphase
Trough
Micro-Flow Imaging
X-Ray Reflectometry
Effect of Surfactant on Air-Water Interface Adsorption
• ~50 Å of dense antibody layer (flat-on)
• Some diffuse layer ~60 Å
• Addition of Triton X-100 (even below CMC) yield significant competition
• Further addition of Triton X-100 yields further depletion of antibody layer
• Addition of more antibody does not negate/reverse effect of surfactant
142 Å 85 Å 38 Å
Head-on End-on Side-on
Flat-on
Air Bulk Solution
PresenterPresentation NotesKnow flat on because not homogenous SLD
Agitation of the Air/Water Interface
Air
Bulk Solution
Air/Water Interface Investigation: Xray Reflectometry
Monolayer Moveable barrier
Subphase
Trough
Micro-Flow Imaging
X-Ray Reflectometry
Air/Water Interface Investigation: Xray Reflectometry
Monolayer Moveable barrier
Subphase
Trough
Micro-Flow Imaging
X-Ray Reflectometry
Micro-Flow Imaging
Flow Cell
Sample
Camera Detection Zone
Examines:
• Size (Equivalent Circular Diameter – ECD)
• Shape (Aspect Ratio)
• Volume (Counts number of particles)
= =
w h
PresenterPresentation NotesAspect ratio - further from one: more elongated
Solid/Water Interface Investigation: Micro-Flow Imaging and Fluorescence Spectroscopy
ThT Fluorescence
Buffer Wash 2 Images:
22.4 uM
30.6 uM
Buffer Wash 2
IgG pH 4.5 Stock Solution
Aspect Ratio
Aspect Ratio
ECD
(uM
) EC
D (u
M)
AU
PresenterPresentation NotesThT binds to elongated, unraveled proteins. Thus, a high peak indicates some elongated particles
3D Histogram MICRONS
Thioflavin T -
Discussion
• Our findings are consistent with earlier findings that surfactants affect protein aggregation and adsorption
• Suggest that surfactants may adsorb to surface of protein itself, or form structure around protein to prevent protein-protein interactions
• Surfactant outcompeted protein at air-water interface and further addition of protein did not negate this effect
Acknowledgments
Thank You to: • Tatiana Perevozchikova • Ron Jones • Katie Weigandt • Prof. Chris Roberts
• Hirsh Nanda • Mike Dimitrou • Bulent Akgun
• SURF Directors • NSF CHRNS • NCNR
Questions?
Thank you.
SANS Data: IgG and Two Surfactants
Solid/Water Interface Investigation: Micro-Flow Imaging and Fluorescence
Flow - in Flow - out
flat Viton spacer
Sample Collection
0
10
20
30
40
50
60
Calculated Rg values: IgG Sample Rg (Å) IgG Stock 49.256 CTAB (from literature) IgG+CTAB above CMC 46.865 IgG+CTAB at CMC 47.504 IgG+CTAB below CMC 48.471 OG (from literature) 23.5 IgG+OG above CMC 40.209 IgG+OG at CMC 46.6 IgG+OG below CMC 47.448 Triton (from literature) 43 IgG+Triton 7.5x CMC 45.079 IgG+Triton 3.75x CMC 45.859 IgG+Triton 2x CMC 45.865 IgG+Triton at CMC 46.459 IgG+Triton below CMC 44.591 Tween (from literature) 26.2 IgG+Tween 2.5x CMC 43.446 IgG+Tween 1.5x CMC 48.496 IgG+Tween at CMC 48.732 IgG+Tween below CMC 44.57 [1] Oliver RC, Lipfert J, Fox DA, lo RH, Doniach S, et al. (2013) Dependence of Micelle Size and Shape on PLoS ONE
8(5): e62488. doi: 10.1371/journal.pone.0062488 [2] Streletzky K, Phillies GDJ. (1994) Temperature Dependence of Triton X-100 Micelle Size and Hydration. Langmuir 11: 42-47 [3] Amani A, York P, de Waard H, Anwar J. (2011) Molecular dynamics simulation of a polysorbate 80 micelle in water. Soft Matter 7: 2900-2908
Rg (Å
)
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