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\begin{document}

\title{Multi Attribute Monitoring Method for Process Development of Engineered
Antibody for Site-Specific Conjugation}



\author[1]{Alistair R. Hines}%
\author[1]{Matthew J. Edgeworth}%
\author[1]{Paul W. A. Devine}%
\author[1]{Samuel Shepherd}%
\author[2]{Nicholas P. Chatterton}%
\author[3]{Claire Turner}%
\author[4]{Kathryn S. Lilley}%
\author[5]{Xiaoyu Chen}%
\author[1]{Nicholas J. Bond}%
\affil[1]{Analytical Sciences Biopharmaceutical Development R\&D AstraZeneca Cambridge UK}%
\affil[2]{The Open University}%
\affil[3]{Brunel University College of Health Medicine and Life Sciences}%
\affil[4]{Cambridge Centre for Proteomics Department of Biochemistry University of Cambridge Cambridge UK}%
\affil[5]{Analytical Sciences Biopharmaceutical Development R\&D AstraZeneca Gaithersburg USA}%


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  \date{May 28, 2024}


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\begin{abstract}
Antibody drug conjugates are a class of biotherapeutic proteins which
have been extensively developed in recent years resulting in new
approvals and improved standard of care for cancer patients. Among the
numerous strategies of conjugating cytotoxic payloads to monoclonal
antibodies, insertion of a cysteine residue at position 239 (C239i)
achieves a tightly controlled, site specific drug to antibody ratio.
Tailored analytical tools are required to direct the development of
processes capable of manufacturing novel antibody scaffolds with the
desired product quality. Here, we describe the development of a high
throughput, 12-minute, mass spectrometry based method capable of
monitoring four distinct quality attributes simultaneously: variations
in the thiol state of the inserted cysteines, N-linked glycosylation,
reduction of inter-chain disulphide bonds, and polypeptide
fragmentation. When deployed, the method provided new insight into the
properties of C239i antibody intermediate and its manufacturing
processes. First, C239i forms exclusively oxidised thiol states within
the bioreactor, of which a variant containing an additional disulphide
bond was invariably produced and remained relatively constant throughout
the fed-batch process; reduced thiol variants were introduced upon
harvest. Second, close to twenty percent of N-linked glycans contained
sialic acid, substantially higher than anticipated for wildtype IgG1.
Lastly, previously unreported polypeptide fragmentation sites were
identified in the C239i constant (C \textsubscript{H}2) domain and the
relationship between fragmentation and glycoform explored. This work
illustrates the utility of applying a high-throughput liquid
chromatography mass spectrometry (LC-MS) multi-attribute monitoring
method to support the development of engineered antibody scaffolds.%
\end{abstract}%



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