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Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy

Antibody‐drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug‐to‐antibody ratio (DAR), which all impact o...

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Autores principales: de Faria e Silva, Ana L., Ryder, Alan G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9828061/
https://www.ncbi.nlm.nih.gov/pubmed/36071600
http://dx.doi.org/10.1002/bit.28229
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author de Faria e Silva, Ana L.
Ryder, Alan G.
author_facet de Faria e Silva, Ana L.
Ryder, Alan G.
author_sort de Faria e Silva, Ana L.
collection PubMed
description Antibody‐drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug‐to‐antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in‐process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing. Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, R (h)) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with R (h) (R (2) = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in‐process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi‐attribute test method for ADC manufacturing.
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spelling pubmed-98280612023-01-10 Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy de Faria e Silva, Ana L. Ryder, Alan G. Biotechnol Bioeng ARTICLES Antibody‐drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug‐to‐antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in‐process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing. Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, R (h)) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with R (h) (R (2) = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in‐process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi‐attribute test method for ADC manufacturing. John Wiley and Sons Inc. 2022-09-28 2022-12 /pmc/articles/PMC9828061/ /pubmed/36071600 http://dx.doi.org/10.1002/bit.28229 Text en © 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle ARTICLES
de Faria e Silva, Ana L.
Ryder, Alan G.
Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title_full Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title_fullStr Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title_full_unstemmed Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title_short Analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
title_sort analyzing protein conjugation reactions for antibody‐drug conjugate synthesis using polarized excitation emission matrix spectroscopy
topic ARTICLES
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9828061/
https://www.ncbi.nlm.nih.gov/pubmed/36071600
http://dx.doi.org/10.1002/bit.28229
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