Pharmaceutical Research

, Volume 28, Issue 12, pp 3128–3144 | Cite as

Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations

  • Xiaoling Wang
  • Sandeep KumarEmail author
  • Satish K. Singh
Research Paper



To explore potential non-canonical disulfide linkages feasible in human IgG2 mAbs via molecular dynamics simulations of a model system, Hinge++.


Hinge++ is derived from the crystal structure of a full-length murine IgG2a antibody by replacing its core hinge region with human IgG2 hinge. Fv and CH3 domains were discarded to speed up calculations. Eight independent simulations, grouped in four sets, were performed. In the control set, disulfide bonding is identical to canonical human IgG2 mAb. Different numbers of disulfide bonds were broken in the remaining three sets.


Two Fabs move towards Fc asymmetrically repeatedly leading to spatial proximity of LC.Cys214 and HC.Cys128 residues in one Fab with Cys residues in the upper hinge region, which could initiate disulfide scrambling. Local dynamics place the eight hinge region Cys residues in a large number of proximal positions which could facilitate non-canonical inter- and intra- heavy chain disulfide linkages in the hinge region.


Consistent with experimental studies, our simulations indicate inter-chain disulfide linkages in human IgG2 mAbs are degenerate. Potential rational design strategies to devise hinge stabilized human IgG2 mAbs are gleaned.


biotherapeutics hinge immunoglobulin molecular modeling structure 



complementarity-determining region


fragment antigen binding


fragment crystallizable


heavy chain


light chain


monoclonal antibody


molecular dynamics


protein data bank


root mean squared deviation



We appreciate the anonymous referees for their constructive criticism of the research work and for suggestions to improve the manuscript. We thank Drs. Sandeep Nema, Sa V Ho, James Carroll, B. Muralidhara, Patrick Buck and Kevin King for several helpful discussions and for critical reading of this manuscript. A postdoctoral fellowship for Xiaoling Wang in Biotherapeutics Pharmaceutical Research and Development, Pfizer Inc. is gratefully acknowledged. High Performance Computing Support received from Pfizer Research Informatics played an essential role in this project.

Supplementary material

11095_2011_503_MOESM1_ESM.doc (286 kb)
Figure S1 Time series of total energy, temperature, volume and time average of pressure of the simulation system for the eight simulations in the production runs. (DOC 286 kb)
11095_2011_503_MOESM2_ESM.doc (54 kb)
Figure S2 The evolution of radius of gyration (Rg) of Hinge++ with simulation time in all trajectories is shown. (DOC 54 kb)
11095_2011_503_MOESM3_ESM.doc (59 kb)
Figure S3 Time courses for (a) sulphur atom distance for the Cys pair LC2.C214-HC2.C236 from the second set of simulations in the four disulfide-bonded conditions described in the manuscript. The solid black circle at time 0 indicates the initial distance; (b). sulphur atom distance for Cys pairs HC2.C128-HC2.C236 from the first set of simulations in the four disulfide-bonded conditions. (DOC 59 kb)
11095_2011_503_MOESM4_ESM.doc (114 kb)
Figure S4 Contact maps for the eight Cys residues in hinge region. (a) Control simulation; (b) Four-reduced2 simulation; (c) Six-reduced2 simulation; (d) All-reduced2 simulation. Grayscale indicates the frequency of observing a given Cys-Cys contact. A contact is defined by sulphur atom distance for two Cys residues being < 5 Å. Note that the order of Cys residues along the X-axes is opposite to that along the Y axes. Hence, these are not traditional contact maps. Each quadrant is labeled according to the nature of contacts. Inter-heavy chain contacts (inter-HC) are located in upper right or lower left quadrants. The two quadrants are symmetric to each other. Intra-heavy chain contacts (Intra-HC1 and Intra-HC2) reside in the rest two quadrants. Each of the two quadrants is symmetric. (DOC 114 kb)
11095_2011_503_MOESM5_ESM.doc (94 kb)
Table S1 Average (μ), standard deviation (σ) and coefficient of variation (ρ=μ/σ) for the overall RMSD profiles in 5 ns simulation time intervals. (DOC 94 kb)
Supplemental Video

The video is from the production run of All-reduced1 simulation. In the video, the sulphur atoms from the six pairs of Cys residues involved in canonical inter-chain disulfide bonds are highlighted as CPK spheres. The sulphur atoms are colored the same as their respective heavy (green and blue) and light (purple and red) chains. Hinge++ molecular model represents only the middle portions of human IgG2 mAbs (see the text for details). During the course of the video, a sulphur atom from a light chain Cys 214 (purple) momentarily moves away from its canonical heavy chain (green) partner and becomes close to other sulphur atoms (green) in upper hinge region. (MPG 18949 kb)


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Xiaoling Wang
    • 1
  • Sandeep Kumar
    • 1
    • 2
    Email author
  • Satish K. Singh
    • 1
  1. 1.Pharmaceutical Research and DevelopmentBioTherapeutics Pharmaceutical Sciences, Pfizer Inc.ChesterfieldUSA
  2. 2.BioTherapeutics Pharmaceutical Sciences Pfizer Inc.St LouisUSA

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