Bioprocess Development and Characterization of a 13 C-Labeled Hybrid Bispecific Antibody Produced in Escherichia coli .
Aaron T WeckslerVictor LundinAmbrose J WilliamsKarthik VeeravalliDorothea E ReillySung-Hye GriecoPublished in: Antibodies (Basel, Switzerland) (2023)
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 13 C-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% 13 C-incorporation using 13 C-glucose and 13 C-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.
Keyphrases
- mass spectrometry
- escherichia coli
- magnetic resonance
- liquid chromatography
- pet imaging
- high resolution
- multiple sclerosis
- ms ms
- small molecule
- blood pressure
- cell therapy
- blood glucose
- metabolic syndrome
- simultaneous determination
- biofilm formation
- staphylococcus aureus
- skeletal muscle
- high resolution mass spectrometry