Gioacchino Luca Losacco

The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time. Herein, we introduce a new automated multicolumn and multi-eluent screening workflow that streamlines the development of new HILIC assays, circumventing the existing tedious ‘hit-or-miss’ approach. A total of 12 complementary columns packed with sub-2 µm fully porous and 2.7 µm superficially porous particles operated on readily available ultra-high pressure liquid chromatography (UHPLC) instrumentation across a diverse set of commercially available polar stationary phases were investigated. Different mobile phases with pH ranging from pH 3 to 9 were evaluated using different organic modifier. The gradient and column re-equilibration were judiciously set to ensure a reliable assay screening framework yielding straightforward separation conditions for subsequent optimization and method deployment in fast-paced laboratory settings. This UHPLC screening system is coupled with a diode array and charged aerosol detectors (DAD and CAD) to ensure versatile detection for a variety of compounds. This fast-screening platform lays the foundation for a convenient generic workflow, accelerating the pace of HILIC method development and transfer across both academic and industrial sectors.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce a hybrid separation technique named Dual‐Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO₂, organic modifier, and water blends with various buffers. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs. After a careful investigation of its fundamental aspects, a DGUC‐DAD‐MS screening workflow that combines multiple orthogonal column and mobile phase choices across a far‐reaching universal elution profile is also reported. The power of this framework is demonstrated with new analytical applications guiding academic and industrial laboratories in the development of new (bio)pharmaceutical targets (e.g. synthetic intermediates, nucleosides, cyclic and linear peptides, proteins, antibody drug conjugates).

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce a hybrid separation technique named Dual‐Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO₂, organic modifier, and water blends with various buffers. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs. After a careful investigation of its fundamental aspects, a DGUC‐DAD‐MS screening workflow that combines multiple orthogonal column and mobile phase choices across a far‐reaching universal elution profile is also reported. The power of this framework is demonstrated with new analytical applications guiding academic and industrial laboratories in the development of new (bio)pharmaceutical targets (e.g. synthetic intermediates, nucleosides, cyclic and linear peptides, proteins, antibody drug conjugates).

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO₂) and deuterated modifiers (CD₃OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH₃OH) and its deuterated counterpart (CD₃OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase’s properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension (¹D) conditions that are combined with MS-friendly RPLC conditions in the second dimension (²D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets. Graphical abstract