(1066-C) Targeted LC-MS with Automated Sample Preparation Enables High-Throughput Screening Assessment of the Conjugatability of SMARTag® Peptides
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: The SMARTag® technology platform offers an efficient chemoenzymatic solution in the creation of site-specific antibody-drug conjugates (ADCs), representing a significant progression in the field of targeted drug delivery systems. One of the important attributes of ADCs is the Drug-Antibody Ratio (DAR), which measures the number of payloads conjugated to the antibody. This method calculates the theoretical maximum DAR for SMARTag antibodies. The objective of this research is to outline a reliable method for determining the maximum DAR in SMARTag antibodies using a formylglycine-generating enzyme (FGE) recognition sequence (LCTPSR), also known as the aldehyde tag. The tag is inserted at the desired location along the antibody backbone using standard molecular biology techniques. Upon expression, FGE catalyzes the conversion of the Cys to a formylglycine residue (fGly), which contains an aldehyde functional group. The aldehyde chemical reactivity is orthogonal to other reactive groups within the antibody sequence and thus serves as a handle to achieve site-specific conjugation. Antibodies carrying aldehyde moieties are reacted with a Hydrazino-iso-Pictet-Spengler (HIPS) linker-payload to generate a site-specifically conjugated ADC. In addition to fGly, the aldehyde tag sequence may undergo modifications that result in the incorporation of glycine (Gly) in place of fGly or a non-specific cleavage at the C-terminus of the Leucine. This process leads to four distinguishable forms of the aldehyde tag sequence, containing either unconverted Cys, fGly, Gly, or a non-specific cleavage, of which, only fGly is conjugatable. Sample preparation to assess fGly content requires denaturing, followed by reduction, Cys derivatization by iodoacetamide, and trypsin digestion. Next, fGly is derivatized using methoxylamine for ease-of-analysis since aldehydes naturally exist in equilibrium with their geminal diol forms. This preparation workflow is substantially streamlined by using an automated liquid handler (Andrew+, Waters™, Andrew Alliance), allowing the preparation and analysis of up to 94 samples, with a standard and blank on a 96-well plate. A dynamic protocol for the Andrew+ was created to automatically adapt this preparation to antibodies of various concentrations from 0.8 to 250 mg/mL. This significantly reduces the preparation and total pipetting time by up to 50%. Moreover, the targeted LC-MS method expedites data collection by up to 75%, allowing for quick data processing. The culmination of this workflow is the calculation of the maximum DAR, incorporating the percentage of fGly, the number of aldehyde tags on the antibody, and the number of payloads per proposed linker-payload structure. To illustrate the practicality of this method, consider a sample where a 95% fGly composition would anticipate a maximum DAR of 1.9, under the assumption of a single payload per linker-payload. In conclusion, this method demonstrates the synergy of automated sample preparation with targeted LC-MS to optimize the screening process of aldehyde tag conjugatability in a high-throughput manner.