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Biopharmaceutical analysis is a rapidly evolving field that requires the development of new technologies and methods to keep pace with the increasing complexity of biologics. One of the most promising areas of research is the use of single-cell omics and microfluidic chips for the analysis of biopharmaceuticals. Single-cell omics has revolutionized our understanding of cellular heterogeneity, while microfluidic chips have enabled high-throughput analysis of single cells that provide an understanding of the complex biological network that complements the genomics and transcriptomics studies. This article will explore some of the emerging trends and technologies in biopharmaceutical analysis, with a particular focus on single-cell omics and microfluidic chips. We will also discuss the developments in ambient ionization mass spectrometry such as sub nanoampere ionization and the potential of low current ionization in studying cell-to-cell heterogeneity and its role in metabolomics., VARIATIONS DISPLAYED by the cell populations in their DNA, RNA, proteins, and metabolites lead to significant heterogeneity and differences in cell states (1). An understanding of the cell-to-cell variations can [...]

Gene therapy is a procedure that enables gene improvement by means of the correction of the altered (mutated) genes, or targets site-specific modifications that have therapeutic treatment as a target [...]

Over the last several years, our definition of biotherapeutics has evolved from solely protein-based therapeutics, such as monoclonal antibodies (mAbs), to include such product classes as cell and gene therapies. Cell and gene therapies are unique products that require innovative and new approaches to formulation, specifically related to drug delivery. One such delivery modality that has gained significant traction is lipid nanoparticles (LNPs), which, as their name suggests, are nanoparticles comprised of lipids. The first LNP product approved by the United States Food and Drug Administration, was the liposome-encapsulated Doxil, whereas, in 2018, the United States Food and Drug Administration approved the small interfering RNA (siRNA) product Onpattro, using lipid nanoparticles as part of its formulation. Since then, LNPs have gained more international attention in their role in drug delivery because of the mRNA-based COVID-19 vaccines. LNPs in the biopharmaceutical space are still in their infancy related to our understanding of them and how to characterize them, presenting both challenges and opportunities. In this column, we discuss some of those challenges and opportunities and look at the current and future landscape of the most common analytical tools used to analyze and characterize LNP, As our understanding of biotherapeutics expands to include new products such as cell and gene therapies, we are presented with new challenges and opportunities in the characterization of these products. [...]

Multi-attribute methods (MAMs) are becoming increasingly popular for their ability to analyze multiple critical quality attributes (CQA) in a single workflow. This capability becomes particularly attractive for a product class [...]

Analytical methods are used in the biopharmaceutical industry to ensure the quality, efficacy, and safety of drug substances and drug products. One of the fundamental measures of the quality of a drug substance or drug product, including biopharmaceuticals, is the stability of the active pharmaceutical ingredient (API). In fact, the first International Council for Harmonization (ICH) guideline on quality, ICH Q1, is for drug stability. In this column, we look at drug stability in general, differences between largeand small-molecule stability (ICH Q1 and ICH Q5), as well as the analytical methods used to measure the stability of a product. Although there are several analytical methodologies that can be used, and we touch on those briefly, we focus mostly on the chromatography and mass spectrometry methods used to characterize drug stability., At the center of everything we do in the biopharmaceutical industry should be patients and ensuring that their medicines are high quality, safe, and effective. A cornerstone of any drug [...]

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

With the number of complex biologics, such as antibody-drug conjugates (ADCs), increasing, reliable, reproducible, and rapid methods are needed for their bioanaly tical charac terization. Arguably, the most common analy tical technologies currently used for characterizing biologics are liquid chromatography (LC) and mass spectrometry (MS) focused on liquid chromatographytandem mass spectrometry (LC-MS/MS) peptide applications. Just as important is the application of LC-MS to analyze the intact mass of these products. In this column, we explore the most current applications of intact mass analysis, such as quantitation, sequencing, and struc tural charac terizing. The advantages and disadvantages of intac t mass analysis are discussed, and a shor t discussion of how peptide characterization is an orthogonal application to intact characterization is presented. The difference between top-down MS and intact mass analysis is explored, because the two terms are often mistakenly used interchangeably. Finally, intact multi-attribute methods (MAMs) are discussed as well as an unique intact mass approach in characterizing ADCs., Characterization of biopharmaceuticals is important throughout the life cycle of the drug product to ensure the safety, efficacy, and quality of the product. This characterizing is particularly important for biologics [...]

Over the last several years, the landscape of the biopharmaceutical industry has begun to change. Monoclonal antibodies (mAbs) still dominate the pipelines in the biopharmaceutical industry; however, more complex molecules, such as antibody-drug conjugates (ADC), are becoming more predominant. In addition to the complexity of ADCs, one of the fastest growing classes of biopharmaceuticals is cell and gene therapies. Gene therapies, for example, use large viral vectors, such as adeno-associated viruses (AAV), as the preferred vector for performing gene therapy. With the complexity of biopharmaceuticals increasing, especially in their size, new and innovative approaches are needed to address and characterize these molecules. This month's column reviews how charge detection mass spectrometry (CDMS) is being used to characterize some of these larger more complex molecules in the biopharmaceutical industry., Biopharmaceuticals are products that are specifically made by living cells or biotechnological processes. By the nature of these products being manufactured in living cells can lead to variability in the [...]

Over the last decade or so, biopharmaceuticals have gained market share and become more complex in their composition. Therefore, new analytical workflows are needed to address these advances, and one [...]

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. [ABSTRACT FROM AUTHOR]

The development of biosurveillance programs with strong analytical performance and economically accessible protocols is essential for monitoring viral pathogens. Throughout the COVID-19 pandemic, whole-genome sequencing (WGS) has been the prevailing technology for SARS-CoV-2 variant of concern (VOC) detection. While WGS offers benefits, it is a lengthy process, financially and technically straining for scalable viral tracking. The aim of this study was to compare the analytical performance and economic feasibility of WGS and PCR mutation panels for distinguishing six known VOCs: Alpha (B.1.1.7 and Q.4), Gamma (P.1), Delta (B.1.617.2 and AY.4.2), and Omicron. (B.1.1.529.1). In all, 78 SARS-CoV-2-positive samples were collected from April to December 2021 at Northeastern University (Cabot Testing Site, Boston, MA, USA) for genotyping PCR and WGS analysis. MagMax Viral/Pathogen II Nucleic Acid Isolation and TaqPath COVID-19 Combo Kits were used for RNA extraction and SARS-CoV-2 confirmation. VOC discrimination was assessed using two TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assay layouts, and Ion Torrent WGS. In November 2021, the mutation panel demonstrated marked versatility by detecting the emerging Omicron variant reported by South Africa. SNP panel analysis yielded the following 78 VOC identifications: Alpha B.1.1.7 ( N = 20), Alpha Q.4 ( N = 3), Gamma P.1 ( N = 1), Delta B.1.617.2 ( N = 30), Delta AY.4.2 ( N = 3), and Omicron B.1.1.529.1 ( N = 20) with one undetermined ( N = 1) sample. Genotyping mutation panels designated lineages in 77 of 78 samples, 46/78 were confirmed by WGS, while 32 samples failed WGS lineage assignment. RT-PCR genotyping panels offer pronounced throughput and sensitivity and provide an economically advantageous technique for SARS-CoV-2 biosurveillance.IMPORTANCEThe results presented in our manuscript demonstrate how the value of simplistic and reliable molecular assays coupled with the core scientific principle of standardization can be overlooked by the charm of more sophisticated assays and instrumentation. This effect can often be amplified during tumultuous public health events, such as the COVID-19 pandemic. By adapting standardized PCR mutation panels to detect prominently circulating SARS-CoV-2 variants, we were able to better assess the potential health impacts of rising positivity rates and transmission clusters within the Northeastern University population. While several literature publications utilizing genotyping PCR and NGS have a similar scope to ours, many investigations lack sufficiently standardized genotyping PCR and NGS bioinformatics inclusionary/exclusionary criteria for SARS-CoV-2 variant identification. Finally, the economic benefits of standardized PCR mutation panels would allow for global implementation of biosurveillance, rather than reserving biosurveillance to more economically developed nations., Competing Interests: The authors declare no conflict of interest.

Background: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area., Methods: We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs., Results: We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections., Conclusions: We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread., Competing Interests: Potential conflicts of interest. M. S. is cofounder of, shareholder in, and advisor to Rhinostics, Inc and reports a patent for an anterior nares swab (US provisional patent application 63/085,571). M. G., D. T., S. R., and E. W. are employed by Integrated DNA Technologies. D. T. reports stock or stock options from Danaher Corporation (payments to author). S. R. reports stock or stock options from Danaher Corporation. C. M. K. is cofounder of Biosens8, Inc and reports grants or contracts unrelated to this work from the NIH National Institute of General Medical Sciences, Defense Advanced Research Projects Agency, Uniformed Services University, and BU; reports consulting fees paid to author from Adventus Research + Consulting, Inc; is a member of the Biomedical Engineering Society Board; and has a leadership or fiduciary role with the American Institute for Medical and Biological Engineering. J. H. C. is a paid consultant for Cell Signaling Technologies and reports funding unrelated to this work from Mass CPR/Evergrande. W. P. H. serves on scientific advisory boards for Biobot Analytics, Inc and Merck; received consulting fees from Biobot Analytics; contributed expert witness testimony on the expected course of the pandemic for Analysis Group; and reports stock or stock options from Biobot Analytics. P. C. S. is a founder of and shareholder in Sherlock Biosciences, reports consulting fees from Sherlock Biosciences, reports patents for CRISPR technology (PCT/US2018/022764 and PCT/US2019/061577), and is on the board and serves as shareholder for the Danaher Corporation. B. A. P. reports lecture honoraria from the Harvard Secondary School Program and a patent for CRISPR technology (WO 2022/051667). D. H. H. reports funding from an institution unrelated to this work (CDC 1 U01CK000632-01-00 GeoSentinel); consultancy payments to the author from Major League Soccer, the Professional Golf Association of America, Equinox, and Xenophon Strategies, Inc; and participation as chair of a data and safety monitoring board for COVEDZ. M. T. G. reports stock or stock options from Danaher Corporation. T. B. reports grants or contracts unrelated to this work from Gilead Sciences, Inc and Fujifilm Pharmaceuticals U.S.A., Inc. M. H. reports consulting fees as an independent laboratory consultant for Nichols Management Group, LLC. All remaining authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.