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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| Published in: | Biotechnology and bioengineering Vol. 119; no. 12; pp. 3432 - 3446 |
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| Main Authors: | , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
Wiley Subscription Services, Inc
01-12-2022
John Wiley and Sons Inc |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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, Rh) 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 Rh (R2 = 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.
Spectroscopic monitoring of antibody drug conjugate (ADC) synthesis can be challenging due to spectral overlap between reactants and products, and because of the need to simultaneously measure multiple process parameters. Here, the authors show how polarized excitation emission matrix (pEEM) can rapidly assess aggregation and quantify the drug to antibody ratio via a single parallel polarized EEM measurement. pEEM is thus a potential process analytical technology for real‐time reaction monitoring of ADC production. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0006-3592 1097-0290 1097-0290 |
| DOI: | 10.1002/bit.28229 |