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High Concentration Formulation Studies of an IgG2 Antibody Using Small Angle X-ray Scattering

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ABSTRACT

Purpose

Concentrated protein formulations are strongly influenced by protein-protein interactions. These can be probed at low protein concentration by e.g. virial coefficients. It was recently suggested that interactions are attractive at short distances and repulsive at longer distances. Measurements at low concentrations mainly sample longer distances, hence may not predict high concentration behavior. Here we demonstrate that small angle X-ray scattering (SAXS) measurements simultaneously collect information on interactions at short and long distances.

Methods

IgG2 antibody samples at concentrations up to 122 mg/ml are analyzed using SAXS and compared to Circular Dichroism (CD), Fluorescence, Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) analysis.

Results

DLS and SEC analyses reveal attraction between antibodies at high concentrations. SAXS data analysis provides an elaborate understanding and shows both attractive and repulsive forces. The protein-protein interactions are strongly affected by excipients. No change in the solution state of IgG2 is observed at pH 4–8, while samples at pH 3 exhibit heavy oligomerization. The solution conformation of the examined IgG2 derived from SAXS data is a T-shape.

Conclusion

SAXS analysis resolves simultaneous attractive and repulsive interactions, and details the effect of excipients on the interactions, while providing three-dimensional structural information from low-concentration samples.

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Abbreviations

+NaCl:

154 mM sodium chloride

+Sucrose:

270 mM sucrose

A2 :

second virial coefficient

BSA:

bovine serum albumine

Buffer only:

no further excipients

CD:

circular dichroism

DLS:

dynamic light scattering

Dmax :

maximal dimension

FF:

form factor

KD :

diffusion virial coefficient

MM:

molecular mass MM

P(r):

pair distance distribution function

Rg :

radius of gyration

Rh :

hydrodynamic radius

SAXS:

small angle x-ray scattering

SEC:

size exclusion chromatography

SF:

the structure factor

SFeff :

effective structure factor

SLS:

static light scattering

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ACKNOWLEDGMENTS & DISCLOSURES

This work was financially supported by Novo Nordisk A/S, the Drug Research Academy, The Danish Council for Independent Research | Medical Sciences and DANSCATT.

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Authors and Affiliations

  1. Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark

    Charlotte Rode Mosbæk & Bente Vestergaard

  2. Novo Nordisk A/S, Måløv, Denmark

    Charlotte Rode Mosbæk & Christian Rischel

  3. European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany

    Petr V. Konarev & Dmitri I. Svergun

Authors
  1. Charlotte Rode Mosbæk
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  2. Petr V. Konarev
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  3. Dmitri I. Svergun
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  4. Christian Rischel
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  5. Bente Vestergaard
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Corresponding author

Correspondence to Bente Vestergaard.

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Mosbæk, C.R., Konarev, P.V., Svergun, D.I. et al. High Concentration Formulation Studies of an IgG2 Antibody Using Small Angle X-ray Scattering. Pharm Res 29, 2225–2235 (2012). https://doi.org/10.1007/s11095-012-0751-3

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  • DOI: https://doi.org/10.1007/s11095-012-0751-3

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