High-resolution two-dimensional liquid chromatography analysis of key linker drug intermediate used in antibody drug conjugates.

In this manuscript, the application of high-resolution sampling (HRS) two-dimensional liquid chromatography (2D-LC) in the detailed analysis of key linker drug intermediate is presented. Using HRS, selected regions of the primary column eluent were transferred to a secondary column with fidelity enabling qualitative and quantitative analysis of linker drugs. The primary column purity of linker drug intermediate ranged from 88.9% to 94.5% and the secondary column purity ranged from 99.6% to 99.9%, showing lot-to-lot variability, significant differences between the three lots, and substantiating the synthetic and analytical challenges of ADCs. Over 15 impurities co-eluting with the linker drug intermediate in the primary dimension were resolved in the secondary dimension. The concentrations of most of these impurities were over three orders of magnitude lower than the linker drug. Effective peak focusing and high-speed secondary column analysis resulted in sharp peaks in the secondary dimension, improving the signal-to-noise ratios. The sensitivity of 2D-LC separation was over five fold better than conventional HPLC separation. The limit of quantitation (LOQ) was less than 0.01%. Many peaks originating from primary dimension were resolved into multiple components in the complementary secondary dimension, demonstrating the complexity of these samples. The 2D-LC was highly reproducible, showing good precision between runs with%RSD of peak areas less than 0.1 for the main component. The absolute difference in the peak areas of impurities less than 0.1% were within ±0.01% and for impurities in the range of 0.1%-0.3%, the absolute difference were ±0.02%, which are comparable to 1D-LC. The overall purity of the linker drug intermediate was determined from the product of primary and secondary column purity (HPLC Purity=%peak area of main component in the primary dimension×%peak area of main component in the secondary dimension). Additionally, the 2D-LC separation enables the determination of potential impurities that could impact the downstream process, like ADCs stability, efficacy and patient safety. Peak capacity of this magnitude, sensitivity and reproducibility of 2D-LC for resolving structurally similar impurities co-eluting with the main component has not been demonstrated to date. This application clearly demonstrates the power of 2D-LC in detailed analysis of structurally similar, co-eluting impurities from key linker drug intermediate used in ADCs that is impossible to achieve by conventional 1D-LC.

Cadherin switching in ovarian cancer progression.

The roles of the cadherins in the progression of ovarian cancer to the late stages of the disease state when malignant cells have disseminated within the peritoneal cavity remain poorly understood. In view of these observations, we have undertaken a comprehensive survey of the cadherin subtypes present in normal ovarian surface epithelium and peritoneum and in the tumors and peritoneal effusions of women diagnosed with Stage I or Stage II primary ovarian cancer using a degenerate cloning strategy for sequences highly conserved among this family of cell adhesion molecules. On the basis of the nucleotide sequences of the resultant PCR products, multiple cadherin subtypes (E-, N-, P-cadherin, and cadherin-4, -6, and -11) were found to be present in these normal and malignant tissues and cells. P-cadherin was determined to be the predominant cadherin subtype in normal peritoneum, peritoneal effusions and Stage II tumor masses. An increase in P-cadherin mRNA and protein expression levels in ovarian tumor masses with progression to later stages of the disease state was confirmed by Northern and Western blot analysis, respectively. In addition, we have determined that the cadherin-associated protein, known as beta-catenin, is expressed in normal peritoneum, ovarian tumors and malignant cell effusions obtained from women with Stage I or Stage II cancer. Immunoprecipitation studies demonstrated that P-cadherin was capable of interacting with beta-catenin in these normal and malignant tissues and cells. Collectively, these findings suggest that the regulated expression of P-cadherin/beta-catenin complexes in ovarian tumor cells may represent a key step in disease progression.