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Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering
Published on: November 5, 2018
High concentration formulation studies of an IgG2 antibody using small angle X-ray scattering
Charlotte Rode Mosbæk1, Petr V Konarev, Dmitri I Svergun
1Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
Pharmaceutical Research
|April 6, 2012
Summary
Small angle X-ray scattering (SAXS) reveals simultaneous attractive and repulsive protein-protein interactions, crucial for understanding concentrated antibody formulations. This method provides structural insights and quantifies excipient effects, unlike low-concentration techniques.
Area of Science:
- Biophysics
- Protein Chemistry
- Formulation Science
Background:
- Concentrated protein formulations are significantly influenced by protein-protein interactions.
- Virial coefficients probe interactions at low concentrations, which may not predict high-concentration behavior.
- Recent hypotheses suggest a combination of attractive short-range and repulsive long-range interactions.
Purpose of the Study:
- To demonstrate that small angle X-ray scattering (SAXS) can simultaneously collect information on protein-protein interactions at both short and long distances.
- To analyze IgG2 antibody samples at high concentrations.
- To compare SAXS findings with other biophysical techniques.
Main Methods:
- Small Angle X-ray Scattering (SAXS) analysis of IgG2 antibody samples up to 122 mg/ml.
- Comparative analysis with Circular Dichroism (CD), Fluorescence, Size Exclusion Chromatography (SEC), and Dynamic Light Scattering (DLS).
Main Results:
- Dynamic Light Scattering (DLS) and SEC indicated antibody attraction at high concentrations.
- SAXS data revealed both attractive and repulsive forces, offering a more comprehensive understanding.
- Excipients significantly affected protein-protein interactions, and pH 3 induced heavy oligomerization of IgG2.
- SAXS determined the solution conformation of IgG2 to be T-shaped.
Conclusions:
- SAXS analysis effectively resolves simultaneous attractive and repulsive interactions in protein formulations.
- SAXS provides detailed insights into the impact of excipients on protein interactions.
- SAXS yields three-dimensional structural information even from low-concentration samples.
