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Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies

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Abstract

Purpose

Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.

Methods

Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments.

Results

Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution.

Conclusion

The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

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Authors and Affiliations

  1. Pharmaceutical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA

    Subhabrata Majumder, Michael Kimmel & Arun Alphonse Ignatius

  2. Analytical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA

    Michael T. Jones

Authors
  1. Subhabrata Majumder
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  2. Michael T. Jones
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Correspondence to Arun Alphonse Ignatius.

Electronic supplementary material

ESM 1

(DOCX 14 kb)

Figure S1

Overlay of 2D [13C-1H] HSQC spectra of mAb-1 Fc (magenta), mAb-2 Fc (red) and mAb-3 Fc (blue). The 2D [13C-1H] NMR spectra of Fc domain shows that the tertiary structure of Fc fragment is nearly identical for all the three mAbs. (PNG 81 kb)

High Resolution (TIF 4802 kb)

Figure S2

Overlay of 2D [13C-1H] HSQC spectra of mAb-1 Fc generated from enzymatic digestion performed on two batches. The near identical spectra shows the robustness of the method. Any changes in crosspeak positions (chemical shift change) are due to chemical changes in local environment due to point mutations.The spectra from batch 1 is indicated in blue and that from batch 2 is indicated in red. (PNG 88 kb)

High Resolution (TIF 350 kb)

Figure S3

SEC-HPLC chromatogram of intact mAb (black), digested mixture (green), purified Fab2 (red) and Fc (blue) domain. The vertical axis indicates UV absorbance and horizontal axis denote elution times. The chromatogram is used to gauge the completion of digestion of enzymatic reaction and to assess purity of the fragments. (PNG 8 kb)

High Resolution (TIF 18 kb)

Figure S4

Mass spectrometric analysis of mAb-2 Fc and mAb-3 Fc. The mass spectra denotes similar glycosylation pattern between the two mAbs Fc. (PNG 56 kb)

High Resolution (TIF 89 kb)

Figure S5

Point mutations of Fc domain can perturb the higher order structure of Fc domain. 2D [15N-1H] HSQC spectra of mAb-3 Fc (yellow) and mAb-2 Fc (blue) indicate distinct chemical shift changes of certain amino acid residues. Each peak corresponds to backbone amide (NH) of different amino acids that make up the Fc domain. The vertical streak in the spectra corresponds to histidine buffer NH peaks. (PNG 169 kb)

High Resolution (TIF 511 kb)

Figure S6

Comparison of a segment of the 2D [13C-1H] NMR spectra of Fc domain of IgG2 mAbs of identical sequence shows molecular fingerprints unique to well behaved mAb. (a) Overlay of mAb-1 Fc and mAb-Control Fc (commercial mAb) shows selected cross peak which are common to both these mAbs marked by rectangle. (b) Overlay of mAb-3 Fc and control mAb Fc shows the absence of the same selected cross peak observed in control mAb Fc but not in mAb-3 Fc. (PNG 64 kb)

High Resolution (TIF 518 kb)

Figure S7

Overlay of a cross section of 2D [13C-1H] spectra of Fc domain of mAb-1 expressed in two different host cells. Differences in post translational modification for the same mAb is not reflected in NMR fingerprinting of Fc in these two cases (blue and red spectrum). (PNG 13 kb)

High Resolution (TIF 183 kb)

Figure S8

Statistical plot of peak amplitudes of mAb-1 Fc, mAb-2 Fc and mAb-3 Fc. (a) Correlation of peak amplitude of 2D [15N-1H] NMR spectrum of mAb-1 Fc and mAb-3 Fc (b) Correlation of peak amplitude of 2D [15N-1H] NMR spectrum of mAb-1 Fc and mAb-2 Fc. (PNG 56 kb)

High Resolution (TIF 335 kb)

Figure S9

Statistical plot of relative peak intensities of mAb-1 Fc, mAb-2 Fc and mAb-3 Fc. The R values suggest higher dissimilarity between mAb-1 Fc and mAb-2 Fc due to point mutation. (a) Correlation of relative peak integrals (intensities) of 2D [13C-1H] NMR spectrum of mAb-1 Fc and mAb-2 Fc (b) Correlation of relative peak integrals (intensities) of 2D [13C -1H] NMR spectrum of mAb-1 Fc and mAb-3 Fc. (PNG 23 kb)

High Resolution (TIF 896 kb)

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Majumder, S., Jones, M.T., Kimmel, M. et al. Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies. Pharm Res 35, 220 (2018). https://doi.org/10.1007/s11095-018-2500-8

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  • DOI: https://doi.org/10.1007/s11095-018-2500-8

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