Antibody Drug Conjugate (ADC) and DAR determination by Dr. Prajita Pandey

Hello everyone.

My name is Prazitha Pandey and I'm Assistant Director Chemistry at Emory Pharma. I have a Bachelor of Science in Biology from University of Texas at arlington and my PhD from University of North Texas. My background spans chromatography, mass spectrometry, proteomics and small molecule analysis. Currently at Emory Pharma I direct projects that involves characterization of antibody drug conjugates, monoclonal antibodies, proteins and peptides at various stages of pharmaceutical development and today I'm here to discuss a very important quality attributes of antibody drug conjugate which is a dart determinations Antibody drug conjugates are.

A type of targeted cancer therapy that.

Combines the specificity of monoclonal antibodies with.

With the cytotoxic effects of chemotherapy drugs. These compounds are designed to deliver a potent cytotoxic payload directly to cancer cells.

While minimizing damage to normal healthy cells.

If you look at the structure of an adc, there is a monoclonal antibody which is the protein that can selectively bind to specific proteins expressed on the surface of cancer cells. The choice of the antibody is crucial as it determines the specificity of the ADC for the cancer cells. The next we have here is a linker which is a chemical bridge connecting the monoclonal antibody to a cytotoxic drug. It is designed to be stable in the bloodstream but can be cleaved or broken down once the ADC has reached the target cell. The cleavage allows the release of the cytotoxic payload inside the cancer cell and and lastly we have a cytotoxic payload which is the potent chemotherapy drug that is conjugated to the antibody via the linker. Once released inside the cancer cell, the cytotoxic payload disrupts cellular processes such as DNA replication or cell division leading to the death of the cancer cell. Drug to antibody determination so what is drug to antibody ratio or DAR and why its determination important? DAR represents the average number of drug molecules conjugated to each antibody molecule within the adc. These conjugation strategies can be categorized based on distinct reactive sites including nonspecific conjugation via native residues, site specific conjugation through genetically engineered site and UV cross linking. In the first example that you see here is a lysine based conjugation which stands out as a highly employed nonspecific conjugation approach. To date, the utilization of lysine chain amines in conjugation technology has advanced into clinical trials incorporating conjugate payloads such as DM1DM4 with monoclonal antibodies. Cysteine based conjugation through cysteine sulfatryl group is another conjugation technology that have progressed into clinical trial. In ongoing clinical trial this conjugation approach is commonly employed to connect MMAE or mmaf. Thirdly, site specific conjugation is an advancement in ADC technology that aims to control the location and number of drug molecules attached to the antibody. Traditional conjugation methods may result in heterogeneous mixtures of ADC with variable dar. Site specific conjugation techniques and aim to overcome this heterogeneity by precisely attaching drug to specific amino acid residues on the antibody. The production of ADCs might result in heterogeneous mixture of an ADC species that consisted of varied DARs. Hence, determining the DAR is crucial for understanding composition and behavior of the ADC as it directly influences the therapeutic efficacy and safety of the drug. Dart Determination Workflow A typical workflow for dart determination using LCHR Ms. Includes sample preparation and based on your sample preparation needs you can incorporate techniques such as ADC enrichment or D glycosylation. Following that is the LC Ms. Analysis where different modes of chromatography such as size exclusion, hydrophobic interaction or reverse phase can be coupled with high resolution mass spectrometry. Then comes the most important part which is the data analysis. Data analysis is accomplished using Biopharma Finder application which calculates a drug load for each components and calculates the average DAR for each raw data file loaded for an experiment. The input for the software is the.

Mass spectra generated from the high resolution.

Mass spectra and the output is the deconvoluted spectra which involves deconvoluting the mass spectra to a series of zero charge masses and then computing average DAR by integrating and weighting the spectral peak area overall. Liquid chromatography coupled with high resolution mass spec is a powerful technique that can determine DAR as it provides precise molecular weight identification of different species ADCs with varying drug conjugation sites or drug payloads, high sensitivity and high throughput analysis. Some of the key details that are required during the data analysis for that determination are sequence information of both heavy chain and light chain, number of disulfide bonds. For example, in an IGG1S there are normally 16 disulfide bonds, 4 interchain and 12 intra chain. As you can see in the figure, glycan composition based on host cell line type and the mass and structure of the linker and payload. Thank you. I hope this was a useful information as it relates to DAR calculation in ADCs. To discuss your project needs, please reach.

Out to us@emorypharma.com or contact us@inforypharma.com thank.

Sam.