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

Authors :
Aaron T. Wecksler
Victor Lundin
Ambrose J. Williams
Karthik Veeravalli
Dorothea E. Reilly
Sung-Hye Grieco
Source :
Antibodies, Vol 12, Iss 1, p 16 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

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.

Details

Language :
English
ISSN :
20734468
Volume :
12
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Antibodies
Publication Type :
Academic Journal
Accession number :
edsdoj.bf85dec8ae9d4900b0494e5eb419cd3b
Document Type :
article
Full Text :
https://doi.org/10.3390/antib12010016