In-Depth Structure and Function Characterization of Heterogeneous Interchain Cysteine-Conjugated Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), integrating high specificity of antigen-targeting antibodies and high potency of cell-killing chemical drugs, have become one of the most rapidly expanding therapeutic biologics in oncology. Although ADCs were widely studied from multiple aspects, overall structural elucidation with comprehensive understanding of variants is scarcely reported. Here, for the first time, we present a holistic and in-depth characterization of an interchain cysteine-conjugated ADC, focusing on conjugation and charge heterogeneity, and in vitro biological activities. Conjugation mapping utilized a bottom-up approach, unraveled positional isomer composition, provided insights into the conjugation process, and elucidated how conjugation affects the physicochemical and biological properties of an ADC. Charge profiling combined bottom-up and top-down approaches to interrogate the origin of charge heterogeneity, its impact on function, and best practice for characterization. Specifically, we pioneered the utilization of capillary isoelectric focusing-mass spectrometry to decode not only critical post-translational modifications but also drug load and positional isomer distribution. The study design provides general guidance for in-depth characterization of ADCs, and the analytical findings in turn benefit the discovery and development of future ADCs.

A simple parallel gas chromatography column screening system.

A simple approach to the automated screening of four different columns on a single gas chromatography (GC) instrument is used for rapid chiral GC method development. Configuration of a conventional GC instrument with a second autosampler and several inexpensive Y-splitters enables simultaneous evaluation of two different columns, allowing a total of four different columns to be evaluated in two automated back to back runs. The resulting system affords a simple and effective approach to chiral GC method development that speeds analysis while eliminating the need for slow and tedious manual interchange of columns. An example of developing a rapid isothermal GC method from the screening results obtained by the instrument is also shown.

Development and validation of ion-pairing HPLC-CAD chromatography for measurement of Islatravir's phosphorylated intermediates.

Biocatalytic processes have become more prevalent in the pharmaceutical industry, leading to analytical challenges not faced when characterizing more traditional synthetic routes. A novel one-pot biocatalytic process has been established for Islatravir, an HIV reverse transcriptase translocation inhibitor for the treatment and prevention of HIV-1. As a one-pot reaction, the Islatravir chemistry contains multiple intermediates that are not isolated. Additionally, these unisolated intermediates have no chromophores, making traditional LC-UV techniques ineffective for characterization. A hydrophilic interaction chromatography (HILIC) method with a charged aerosol detector (CAD) was initially developed, however numerous inorganic species present in the one-pot reaction were retained; this led to co-elution of compounds and poor peak shapes. An innovative ion-pairing LC method was developed in order to resolve inorganic species, intermediates, and the API, for use during in-process control of the Islatravir biocatalytic reaction. Aided by a volatile ion-pairing reagent compatible with the CAD, this method successfully retains and resolves the highly polar intermediates of interest and Islatravir API. This novel method was successfully validated and has allowed the Islatravir biocatalytic process to be fully characterized from the early intermediates through the final API within the one-pot reaction without the need for isolations. This novel ion-pairing HPLC-CAD technique lays the groundwork for method development on current and future biocatalytic-produced drug substances.

A miniature mass spectrometer for liquid chromatography applications.

A miniature mass spectrometer capable of detecting analytes eluting from a high-performance liquid chromatography (HPLC) system is described and demonstrated for the first time. The entire instrument, including all pumps and the computer, is contained within a single enclosure that may be conveniently accommodated at the base of the HPLC stack. The microspray ion source, vacuum interface, ion guide, and quadrupole ion filter are all microengineered. These components are fabricated in batches using microelectromechanical systems (MEMS) techniques and considered to be consumables. When coupled to a standard HPLC system using an integrated passive split, the limit of detection for reserpine while scanning the full mass range is 5 ng on-column (1 pg of which is passed to the microspray). The mass range is m/z 100-800, and each spectrum is typically acquired at a rate of 1 scan per second.