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.

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.

Selection of column and mobile phase eluent/buffer is a crucial step in chromatographic method development. The introduction of new column technologies requires a continuous performance evaluation enabling the deployment of improved chromatographic screening workflows. Consequently, the development of a universal method that could be used across different column chemistries and mobile phase pH ranges would be highlighted as advantageous. Herein, we introduce a single generic probe developed via software-based modeling that delivers excellent results for the separation of a standard multicomponent mixture across a wide range of stationary phases under different pH and temperature conditions. This approach has been applied to interrogate the performance and stability of multiple columns (C18, Cyano, Phenyl Hexyl, PFP, Polar-embeded, and other stationary phases) using an automated ultra-high-pressure liquid chromatography screening system illustrating its practicality and effectiveness.

Online mass spectrometry has proven to be a useful tool for characterizing many aspects of chemical reactions. However, to the best of the authors' knowledge, no reference standard (RS) quantitation approach has been applied in online MS profiling work to date. In this study, we present a RS approach for online quantitation of an aerobic oxidation reaction in flow using a miniature mass spectrometer, with both internal RS and external RS quantitation approaches being evaluated. Quinoline, a structurally similar and chemically inert compound under these reaction conditions, was chosen as the RS to quantify the pyridine aldehyde product. To investigate the optimal RS concentration and instrument attenuation, calibration curves were established by plotting the product/RS peak intensity ratio against the theoretical product yield at different attenuation (dilution factor) values. The MS quantitation results for the actual flow reactions were validated with conventional offline H-1 NMR analysis.

Liters of organic solvents are stored in analytical laboratories across the pharmaceutical industry for the sole purpose of preparing process solvent standards that contain only microliters of solvent. The accumulation of a 10(6)-fold excess volume of unnecessary solvent is a dubious practice from both an environmental and a safety perspective. The Enabling Technologies Consortium (ETC), comprised of international pharmaceutical companies, has explored the use of pre-made solvent standards in 1 mL ampules to circumvent the environmental and safety concerns created by this common practice. In the reported work, two ampules of pre-made standards containing 29 of the most commonly used solvents in pharmaceutical development and manufacturing were evaluated. Creating mixtures of chemically compatible standards as opposed to individual standards for each solvent further minimizes the need to maintain a large inventory of multiple different ampules. Pre-made standard mixtures in conjunction with generic gas chromatography-flame ionization (GC-FID) methods, were evaluated against in-house standards of each solvent prepared according to the different GMP protocols at each company. Comparability was excellent for all solvents tested, as indicated by an overall recovery of 100.1 +/- 2.8%. These pre-made standard ampules also provide numerous advantages from both speed and greenness standpoints, including a much faster turnaround time of analytical results, with significantly less labor and greater reliability. In addition, this ETC effort demonstrates it is possible to maintain superior quality control of residual solvents in drug substances while improving safety and efficiency across the industry, as well as reducing the environmental footprint to perform the necessary experiments to ensure patients safety.

A survey of different strategies for chromatographic method development in pharmaceutical research and development is presented. Owing to the widespread utilization of chromatography within diverse areas of pharmaceutical research, a variety of strategies for method development have arisen. We survey the current state of the art, discuss recent trends and approaches and highlight future prospects and capability gaps.

The use of gas chromatography with headspace sampling is commonplace in analytical laboratories for the analysis of residual solvents. In this article, we discuss the use of an internal standard-based calibration, utilizing relative response factors, to enable the generation of accurate weight % solvent data on 25 common solvents in a single chromatographic run. The total cycle time for analysis is less than 30 min. To facilitate this technology into the process chemistry environment, bespoke open access software has been developed to simplify the sample submission process such that virtually no training is required to analyze the sample, process the data, and generate a report. Furthermore, an automated calibration check workflow has been implemented to validate the quality of the data on a daily basis and alert the system administrator in the event of a problem with the system.

An overview of recent progress in the development of compact mass spectrometers for use as chromatographic detectors in chemical analysis is presented. As the applications of LC-MS technologies have grown in recent years there has been a continued expansion of the approach to new user groups. Within the pharmaceutical industry, the recent development of small, inexpensive and quiet MS detectors for LC has enabled the rollout of this important technology well beyond the initial user base of researchers in drug metabolism and bioanalysis to the direct support of research areas such as discovery chemistry, process chemistry, chemical engineering, manufacturing and formulation sciences, with comparable broadening of the MS user base occurring in other industries and in academia. In this review we survey recent developments and applications ranging from reaction monitoring, biomolecule analysis and high throughput microplate analysis to the identification and analysis of impurities, degradation products and potential mutagens, offering thoughts on current limitations and future directions. (C) 2016 Elsevier B.V. All rights reserved.

In this study we describe the evaluation of a recently developed miniaturized single-quadrupole mass spectrometer to support pharmaceutical process research investigations. Mass spectrometry is becoming an indispensable tool for analytical support of synthetic chemistry; however, current mass spectrometers are too expensive and too large for widespread deployment. In addition, current instruments often have features and capabilities that, while useful for trace component or bioanalysis applications, are beyond the comparatively simple requirements of synthetic chemists, where samples are often abundant and unit mass resolution is generally sufficient. An evaluation of the Microsaic 3500 MiD shows this small and inexpensive mass spectrometer to be well-suited for providing reliable support for certain pharmaceutical process research investigations.

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.

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.

In this chapter, the pursuits of the modern analytical laboratory, supporting pharmaceutical process development, will be discussed. The field of pharmaceutical process development is an ever changing landscape. As the drive to reduce cycle time increases, the need to gain as much information as possible about a process, and fully exploit the available data generated, becomes more critical. Analytical chemistry is constantly evolving to address the changes in paradigm and provide more detailed assessment of reaction kinetics and impurity profiling. The ability to develop analytical methodology in hours, rather than days, has enabled the synthetic chemist to move from fume hood to pilot plant more rapidly, while the use of on-line analytical tools at the location of the reaction, collecting data in real time, has provided greater confidence in scaling of reactions. Reductions in cycle times cannot occur at the expense of quality, and the need for validated methodology to support agency regulations has not relaxed. The synergy between process and analytical chemistry grows ever stronger, and the importance of a flexible interface between analytical and process becomes even more critical in addressing the associated challenges. The analytical function is no longer just a confirmation of the desired product and assurance of quality, but also a critical support function, guiding all aspects of the synthetic endeavour. The valuable information generated in well planned analytical experiments not only assists the process chemist in developing a more efficient process which impacts the overall cost of goods, but also impacts operator and ultimately the patient safety.

A highly convergent synthesis of c-Met kinase inhibitor 1 has been demonstrated on a multikilogram scale using three key fragments: dihalotricyclic core 2, chiral sulfamide side chain 3, and pyrazole boronic ester 4. The chirality in sulfamide side chain 3 was installed using the cheap and readily available starting material (S)-epichlorohydrin. A total of 2.71 kg of 1 were isolated in seven steps (the longest linear sequence).

The development of a synthetically useful, regioselective cross-coupling of 2,4-diaminopyridines with aryl and heteroaryl halides is reported. Selectivity for coupling through either amine is controlled by a simple change in the reaction conditions. Cross-coupling through the 2-amino group predominates in the presence of a palladium catalyst, whilst the 4-amino coupled product predominates in the absence of palladium. (C) 2009 Elsevier Ltd. All rights reserved.