# A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization

## TL;DR

This paper introduces a new bioconjugation method to create immunoconjugates with better control and a new workflow to analyze their structure using mass spectrometry.

## Contribution

A kinetically controlled bioconjugation method and a domain mapping MS-workflow for immunoconjugate characterization are developed.

## Key findings

- A kinetically controlled reaction achieved intradomain regioselective bioconjugation of Trastuzumab with DOTA.
- LC–MS and domain mapping revealed specific lysine residues and domains involved in conjugation.
- The immunoconjugate mixture contained 15 species, with DOTA distribution quantified across antibody domains.

## Abstract

Immunoconjugates exploit the high affinity of monoclonal
antibodies
for a recognized antigen to selectively deliver a cytotoxic payload,
such as drugs or radioactive nuclides, at the site of disease. Despite
numerous techniques have been recently developed for site-selective
bioconjugations of protein structures, reaction of ε-amine group
of lysine residues with electrophilic reactants, such as activated
esters (NHS), is the main method reported in the literature as it
maintains proteins in their native conformation. Since antibodies
hold a high number of lysine residues, a heterogeneous mixture of
conjugates will be generated, which can result in decreased target
affinity. Here, we report an intradomain regioselective bioconjugation
between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic
acid (DOTA) by a kinetically controlled reaction adding substoichiometric
quantities of the activated ester to the mAb working at slightly basic
pH. Liquid chromatography–mass spectrometry (LC–MS)
analyses were carried out to assess the chelator-antibody ratio (CAR)
and the number of chelating moieties linked to the mAb chains. Proteolysis
experiments showed four lysine residues mainly involved in bioconjugation
(K188 for the light chain and K30, K293, and K417 for the heavy chain),
each of which was located in a different domain. Since the displayed
intradomain regioselectivity, a domain mapping MS-workflow, based
on a selective domain denaturation, was developed to quantify the
percentage of chelator linked to each mAb domain. The resulting immunoconjugate
mixture showed an average CAR of 0.9. About a third of the heavy chains
were found as monoconjugated, whereas conjugation of the chelator
in the light chain was negligible. Domain mapping showed the CH3 domain
bearing 13% of conjugated DOTA, followed by CH2 and VH respectively
bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found
in the CH1 domain, whereas for the light chain, only the CL domain
was conjugated (6%). Data analysis based on LC–MS quantification
of different analytical levels (intact, reduced chains, and domains)
provided the immunoconjugate formulation. A mixture of immunoconjugates
restricted to 15 species was obtained, and the percentage of each
one within the mixture was calculated. In particular, species bearing
1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison
to species bearing 2DOTA, were observed. Pairing of bioconjugation
under kinetic control with the developed domain mapping MS-workflow
could raise the standard of chemical quality for immunoconjugates
obtained with commercially available reactants.