Ion-pair reversed-phase and hydrophilic interaction chromatography methods for analysis of phosphorothioate oligonucleotides.

In this study we investigated the separation of a 25 mer fully phosphorothioated oligonucleotide from its truncated n-1 (24 mer) species and selected phosphodiester 25 mer impurities using ion-pair reversed-phase chromatography. The hydrophobicity of ion-pairing agents (alkylamines) impacts n-1 separation selectivity. 25 mer impurities with single and double phosphodiester bonds eluted prior to the parent phosphorothioate oligonucleotide in the same region as 24 mer impurities, which complicated the chromatographic separation.

Monitoring stability indicating impurities and aldehyde content in lipid nanoparticle raw material and formulated drugs.

Lipid nanoparticles (LNPs) are designed to protect and transport sensitive payloads or active pharmaceutical ingredients as part of new therapeutic modalities. As a multi-component particle, a high degree of quality control is necessary to ensure raw materials are free of critical impurities that could adversely impact the drug product. In this study, we demonstrate a reversed phase liquid chromatography method hyphenated with a single quadrupole mass spectrometer (RPLC-MS) as an alternative platform to methods that incorporate evaporative light scattering or charged aerosol detectors in the detection and quantitation of critical impurities associated with LNPs.

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals.

Lipid encapsulated messenger RNA (LNP mRNA) has garnered a significant amount of interest from the pharmaceutical industry and general public alike. This attention has been catalyzed by the clinical success of LNP mRNA for SARS-CoV-2 vaccination as well as future promises that might be fulfilled by the biotechnology pipeline, such as the in vivo delivery of a CRISPR/Cas9 complex that can edit patient cells to reduce levels of low-density lipoprotein. LNP mRNAs are comprised of various chemically diverse molecules brought together in a sophisticated intermolecular complex.

Reducing metal-ion mediated adsorption of acidic peptides in RPLC-based assays using hybrid silica chromatographic surfaces.

In this study we evaluate column hardware exhibiting a novel hybrid silica surface in its ability to mitigate metal-induced adsorption artifacts such as chromatographic peak tailing for acidic amino acid residue containing peptides. Using a conventional reversed-phase liquid chromatography (RPLC)-based method, chromatographic performance of a peptide map was compared using a traditional stainless-steel column and an equivalent column bearing a novel hybrid silica surface. Tailing factors for six peptides containing acidic amino acid residues (T ≥ 1.

High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated LC-MS-Based Solution.

Protein glycosylation can impact the efficacy and safety of biotherapeutics and therefore needs to be well characterized and monitored throughout the drug product life cycle. Glycosylation is commonly assessed by fluorescent labeling of released glycans, which provides comprehensive information of the glycoprofile but can be resource-intensive regarding sample preparation, data acquisition, and data analysis. In this work, we evaluate a comprehensive solution from sample preparation to data reporting using a liquid chromatography-mass spectrometry (LC-MS)-based analytical platform for increased productivity in released glycan analysis.

Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides.

Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention.

Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays.

Metal-ion mediated adsorption in liquid chromatography has been identified as a contributing factor in poor peak shape, tailing, and diminished recovery of compounds prone to cation exchange-like interaction with metal-based activity sites. Peptides that exhibit negative charge-bearing amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal-ion mediated adsorption in RPLC/MS-based separations when using weak acids (e.g.

[The Characterization of Drug-to-Antibody Ratio and Isoforms of ADCs Using LC and MS Technologies].

Hydrophobic interaction chromatography (HIC) and MS are leading techniques for the characterization of the critical quality attributes (CQA) of antibody-drug conjugates (ADCs). This includes the average drug-to-antibody ratio (DAR) and drug loading distribution. A workflow that effectively utilizes the synergy between chromatography and detection technologies has been developed and was assessed using cysteine-conjugated ADCs.

Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis.

Rationale: Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI-MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation.

A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window.

A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography.

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC).

RPLC of intact proteins using sub-0.5 μm particles and commercial instrumentation.

This paper addresses whether one can gain an improvement in speed or resolution with a silica colloidal crystal (SCC) of nonporous 470 nm particles when using a commercial nano-UHPLC. Compared to a capillary packed with nonporous 1.3 μm particles and the same C4 bonded phase, the peak width for BSA is decreased by a factor of 6.

Silica colloidal crystals as emerging materials for high-throughput protein electrophoresis.

Silica colloidal crystals are a new type of media for protein electrophoresis, and they are assessed for their promise in rapidly measuring aggregation of monoclonal antibodies. The nature of silica colloidal crystals is described in the context of the need for a high-throughput separation tool for optimizing the formulations of protein drugs for minimal aggregation. The fundamental relations between molecular weight and mobility in electrophoresis are used to make a theoretical comparison of selectivity between gels and colloidal crystals.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide.

Sieving of proteins in silica colloidal crystals of millimeter dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data.

Effects of lead and mercury on the blood proteome of children.

Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry.