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Client
Australian University
Sector
Research
Research Domain
Immuno-Oncology
Application
Ovarian cancer
Key processes
The project aimed to generate a characterized ADC for an academic preclinical proof-of-concept study in ovarian cancer immuno-oncology. Using the client’s full-length human IgG1 antibody and selected Vc-MMAE payload, ProteoGenix developed a site-specific conjugation workflow targeting a DAR of 4 while preserving antibody integrity.
The development strategy was organized in two sequential phases. First, a feasibility study was performed to validate the experimental conditions and identify the best conjugation strategy. Second, the selected condition was transferred to a larger-scale coupling and characterization phase.
The final objective was to obtain a monoclonal antibody coupled to Vc-MMAE with a controlled DAR of 4 while preserving antibody integrity and limiting aggregation.
Establishing native antibody suitability for site-specific ADC conjugation
Achieving controlled conjugation to Vc-MMAE with a target DAR of 4
Maintaining antibody monomeric purity after the bioconjugation process
Preserving antibody integrity before and after conjugation
ProteoGenix implemented a two-step ADC development workflow, starting with a feasibility study to validate the conjugation conditions, followed by larger-scale coupling and characterization.
The native full-length human IgG1 antibody was first analyzed by HPLC-HIC, HPLC-SEC, and SDS-PAGE to confirm its suitability for conjugation. These analyses established the DAR 0 reference profile, confirmed that the antibody was predominantly monomeric, and showed no detectable fragmentation.
The antibody was buffer-exchanged into an appropriate phosphate buffer and conjugated to Vc-MMAE using a site-specific strategy based on a MC-Val-Cit-PAB linker. Several conditions were tested during the feasibility phase to identify the most suitable conjugation parameters.
The resulting ADC was characterized by HPLC-HIC, HPLC-SEC, and SDS-PAGE to assess DAR distribution, monomeric purity, aggregation, and antibody integrity. The best-performing condition was then applied to a larger-scale conjugation, using the same analytical workflow to confirm reproducibility and final ADC quality.
The feasibility phase confirmed that the customer-provided full-length human IgG1 antibody was suitable for site-specific conjugation to Vc-MMAE. The native antibody was predominantly monomeric, showed no detectable fragmentation by SDS-PAGE, and displayed a consistent HPLC-HIC retention time corresponding to DAR 0.
Following conjugation, HPLC-HIC demonstrated that the target DAR 4 species was predominant, with an average DAR of 4.0. SEC analysis confirmed that the conjugation process did not induce detectable aggregation, with a monomeric purity of 96.1%.
The larger-scale conjugation reproduced the feasibility results with a similar HPLC-HIC profile. As shown in the figure, the final ADC batch displayed a predominant DAR 4 species, representing 56.7% of the relative peak area, with an average DAR of 3.8. SEC analysis confirmed the absence of aggregation after conjugation, with a monomeric purity of 92.2%.
A little over 6 mg of final ADC was delivered to the client, together with a complete analytical report. This provided the academic team with a characterized ADC batch and the supporting data needed to continue their preclinical proof-of-concept evaluation in ovarian cancer immuno-oncology.
Together, these analytical results confirmed that the site-specific conjugation strategy enabled controlled coupling of the full-length human IgG1 antibody to Vc-MMAE while preserving antibody integrity and maintaining high monomeric purity.
ProteoGenix developed a site-specific ADC conjugation workflow for a full-length human IgG1 antibody coupled to Vc-MMAE, supporting an ovarian cancer preclinical proof-of-concept project.
By combining feasibility testing, controlled conjugation, and analytical validation, ProteoGenix helped reduce development risk while addressing the project’s main challenges: confirming antibody suitability, achieving a DAR close to 4, and preserving ADC quality after conjugation.
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