The Creation of an Antibody Drug Conjugate, T-mab-VC-Dox

Undergraduate #154
Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)

Zoe Vaughn - University at Buffalo
Co-Author(s): Alexandra Van Hall, David Bussing, and Dhavalkumar Shah, University at Buffalo, Buffalo, NY

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ADCs are a biotherapeutic treatment that consists of a monoclonal antibody, linker, and cytotoxic drug [1]. We propose the linker, MC-val-cit-PAB-pNP, and drug, doxorubicin, conjugation will result in a payload, linker and drug, suitable for attachment to an antibody, trastuzumab, resulting in an ADC. The reaction product is purified then is identified by TLC and Mass Spectrometry. The experiment showed an m/z ratio of the conjugate at 1142. Through DPBS buffer exchange t-mab is brought to a 4.97 mg/ml concentration. TCEP a reducing agent, used to find the free thiol and to estimate the drug antibody ratio, DAR. T-mab and payload incubated together to allow conjugation to occur. The ADC is then eluted and pooled to be tested through size exclusion and HIC. The size exclusion experiment showed the same peaks for t-mab and VC-Dox. HIC experiment showed a DAR of around 2 in HIC. The desire DAR of 4 was not obtained because method used not optimized for T-mab-VC-dox, ADC, the ADC will be tested with a papain experiment to de-conjugate the Dox to determine the true DAR of the ADC.

Funder Acknowledgement(s): Alexandra Van Hall and Sharma Sharad

Faculty Advisor: Dhavalkumar Shah, dshah4@buffalo.edu

Role: I conduced all of the experiments, synthesizing the linker. Analyzing the VC-dox product on TLC plates, then on the HPLC. From there conducting the antibody conjugation, conducting that experiment in its entirety. I was assisted with analysis with HIC and size exclusion. Finally creating the papain standard curve and conducting that experiment I did myself. I was assisted by a doctoral student in the lab. For the most part it was my own project.