Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

MAbs. 2018 Oct;10(7):992-1002. doi: 10.1080/19420862.2018.1496879. Epub 2018 Jul 30.

Abstract

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

Keywords: Deuterium labeling; monoclonal antibody; nuclear magnetic resonance (NMR); protein labeling; stable isotope labeling.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / therapeutic use
  • Biological Therapy*
  • Carbon Isotopes / chemistry
  • Escherichia coli / genetics*
  • Gene Expression
  • Glycosylation
  • Humans
  • Immunosorbent Techniques
  • Isotope Labeling / methods*
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry

Substances

  • Antibodies, Monoclonal
  • Carbon Isotopes
  • Carbon-13

Grant support

This work was supported by the National Institute of Standards and Technology (NIST).