Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

Utility of bacterial peptidoglycan recycling enzymes in the chemoenzymatic synthesis of valuable UDP sugar substrates.

Uridine diphosphate (UDP) sugars are essential precursors for glycosylation reactions in all forms of life. Reactions that transfer the carbohydrate from the UDP donor are catalyzed by glycosyltransferases (Gtfs). While the stereochemistry and negative physiological charge of UDP-sugars are essential for their biochemical function in the cell, these characteristics make them challenging molecules to synthesize and purify on scale in the laboratory. This chapter focuses on the utilization of a chemoenzymatic synthesis of muramyl UDP-sugars, key building blocks in the bacterial cell peptidoglycan. A scalable strategy to obtain UDP-N-acetyl muramic acid derivatives (UDP-NAM), the first committed intermediate used solely in peptidoglycan biosynthesis, is described herein. This methodology utilizes two enzymes involving the cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1-phosphate uridylyl transferase (MurU), respectively. The promiscuity of these enzymes allows for the unique chemical functionality to be embedded in bacterial peptidoglycan both in vitro and in whole bacterial cells for subsequent structural and functional studies of this important biopolymer.

Modulation of the NOD-like receptors NOD1 and NOD2: A chemist's perspective.

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.

Determination of the binding affinities of Neisseria meningitidis serogroup W capsule polymerase with two nucleotide sugar substrates.

OBJECTIVE: Meningococcal meningitis is a public health burden. Immunization strategies have reduced global incidence of the disease. Glycoconjugate vaccines are the most effective type of vaccine to combat most causes of meningococcal meningitis. These vaccines contain capsular polysaccharide fragments from disease-causing serogroups of Neisseria meningitidis that are chemically attached to a carrier protein. The enzymes responsible for capsular polysaccharide synthesis can serve as tools to make these critical vaccine components. One such enzyme is the N. meningitidis serogroup W capsule polymerase. This enzyme is responsible for creating the galactose-sialic acid containing capsular polysaccharide of this serogroup. Our aim in this study was to determine the binding affinities of nucleotide sugar donors CMP-sialic acid and UDP-galactose using a coupled transferase assay to inform future work to modulate polysaccharide synthesis by this enzyme. RESULTS: We determined a Km of 66.8 µM for CMP-sialic acid and a Km for UDP-galactose of 3.9 µM. These values are lower than reported values for other retaining galactosyltransferases and inverting sialyltransferases respectively. There were difficulties obtaining reliable data for galactosyltransferase activity. An alternate strategy is needed to assess kinetic parameters of the separate transferase activities for this enzyme.