An Investigation of the Stabilizing Effects of Surfactants ... · [2] Streletzky K, Phillies GDJ....

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)


Protein Adsorption (one of the possible configurations) at air-water interface

LAir

LHead Groups LSolvent

LProtein

LBulk Solvent

LTails

Langmuir Trough Work:


1 mg/ml IgG

+Triton X-100 (below CMC)

+Triton X-100 (above CMC)

+IgG (5mg/ml)

Dilate/Compress Barrier

???

Competitive Adsorption


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


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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An Investigation of the Stabilizing Effects of Surfactants ... · [2] Streletzky K, Phillies GDJ....

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