Bioprocess Development and Characterization of a 13C-Labeled Hybrid Bispecific Antibody Produced in Escherichia coli

Abstract

Monoclonal antibodies (mAbs) are highly efficacious therapeutics; however, due to their large, dynamic nature, structural perturbations and regional modifications are often difficult to study. Moreover, the homodimeric, symmetrical nature of mAbs makes it difficult to elucidate which heavy chain (HC)-light chain (LC) pairs are responsible for any structural changes, stability concerns, and/or site-specific modifications. Isotopic labeling is an attractive means for selectively incorporating atoms with known mass differences to enable identification/monitoring using techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR). However, the isotopic incorporation of atoms into proteins is typically incomplete. Here we present a strategy for incorporating ¹³C-labeling of half antibodies using an Escherichia coli fermentation system. Unlike previous attempts to generate isotopically labeled mAbs, we provide an industry-relevant, high cell density process that yielded >99% ¹³C-incorporation using ¹³C-glucose and ¹³C-celtone. The isotopic incorporation was performed on a half antibody designed with knob-into-hole technology to enable assembly with its native (naturally abundant) counterpart to generate a hybrid bispecific (BsAb) molecule. This work is intended to provide a framework for producing full-length antibodies, of which half are isotopically labeled, in order to study the individual HC-LC pairs.

Keywords: 13C-celtone; 13C-labeled glucose; ambr250 bioreactors; biotherapeutic proteins; knob-into-hole; protein isotopic labeling.

Figure 1

End-of-run growth (A) and initial osmolality (B) of E. coli cultures expressing hAb1 grown in primary inoculum culture media containing tryptone and yeast extract (control), unlabeled Celtone (Celtone), and unlabeled Celtone 2-fold dilution (Celtone 2-fold). Results shown are from n = 1 experiment. Growth (C) and osmolality (D) of E. coli cultures expressing hAb1 grown in production fermentation media containing NZ Soy BL4 and yeast extract (filled circles), unlabeled Celtone (open circles), unlabeled Celtone 2-fold dilution (open triangles), and unlabeled Celtone 4-fold dilution (open diamonds). Results shown are from n = 1 experiment.

Figure 2

Growth (A), osmolality (B), titer (C), and phosphate depletion (D) of E. coli cultures expressing hAb1 grown in production fermentation media containing NZ Soy BL4 and yeast extract (filled circles), unlabeled Celtone 4-fold dilution (filled diamonds), and E. coli cultures expressing hAb2 grown in media containing ¹³C-labeled Celtone 4-fold dilution (filled squares). Results shown are from n = 1 experiment. Note: ¹³C-labeled fermentation was terminated at 64 h due to operational challenges.

Figure 3

Intact mass analysis of the hybrid molecule (right side) compared to the predicted WT molecule (left side).