Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations.

The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.

Development of an ultra-high-performance liquid chromatography-charged aerosol detection/UV method for the quantitation of linear polyethylenimines in oligonucleotide polyplexes.

Linear polyethylenimines are polycationic excipients that have found many pharmaceutical applications, including as a delivery vehicle for gene therapy through formation of polyplexes with oligonucleotides. Accurate quantitation of linear polyethylenimines in both starting solution and formulation containing oligonucleotide/polyethylenimine polyplexes is critical. Existing methods using spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry time-of-flight, or nuclear magnetic resonance are either complex or suffer from low selectivity. Here, the development and performance of a simple analytical method is described whereby linear polyethylenimines are resolved by ultra-high-performance liquid chromatography and quantified using either a charged aerosol detector or an ultraviolet detector. For formulated oligonucleotide/polyethylenimine polyplexes, sample preparation through decomplexation/digestion by trifluoroacetic acid was necessary to eliminate separation interference. The method can be used not only to support formulation development but also to monitor the synthesis/purification and characterization of linear polyethylenimines.

Accelerated Forced Degradation of Therapeutic Peptides in Levitated Microdroplets.

PURPOSE: Forced degradation is critical to probe the stabilities and chemical reactivities of therapeutic peptides. Typically performed in bulk followed by LC-UV or LC-MS analysis, this traditional workflow consists of a reaction/analysis sequence and usually requires half a day to several days to form and measure the desired amounts of degradants. A faster method is needed to study peptide degradation in a shorter time in order to speed up the drug development process. METHODS: In the new rapid method developed in this study, peptide degradation occurs in levitated aqueous microdroplets using the Leidenfrost effect. RESULTS: This two-minute reaction/analysis workflow allows major degradation pathways of Buserelin, Octreotide, Desmopressin and Leuprorelin to be studied. The reactions include deamidation, disulfide bond cleavage, ether cleavage, peptide bond hydrolysis, and oxidation. CONCLUSIONS: The accelerated forced degradation method requires a minimal amount of therapeutic peptide per stress condition, and the appropriate extent of degradation can be readily generated in seconds by adjusting the droplet levitation time. Levitated microdroplets should be applicable in pharmaceutical development to rapidly determine the intrinsic stability of therapeutic peptides and to aid formulation development by screening the effects of excipients on the stability of the peptides. Graphical abstract.

Development of an automated and High throughput UHPLC/MS based workflow for cleaning verification of potent compounds in the pharmaceutical manufacturing environment.

Cleaning verification (CV) is a critical step in the pharmaceutical manufacturing process to eliminate or reduce unacceptable contamination of a product as a result of insufficiently cleaned equipment surfaces. The main concern is cross contamination with active pharmaceutical ingredients (APIs) from previous runs that may impact patient safety. Current conventional approaches involve rather tedious sample preparation and analytical methods with relative lengthy analysis time. Potent APIs possessing low acceptable daily intake (ADI) values require analytical methods for CV with very low detection limits to confirm that these APIs are below their acceptance limits prior to the next manufacturing process. In this work, a novel end to end CV workflow was developed, which includes the automated sample and calibration solution preparation as well as high throughput analysis by ultra-high-performance liquid chromatography (UHPLC) coupled with single quadrupole mass spectrometry in multiple injection chromatography and selected ion monitoring mode (MIC-MS-SIM). The method was validated using ten model compounds. Acceptable specificity, linearity (R2 > 0.997) and single digit ng/mL LOQ and LOD were achieved for all model compounds. This approach was also successfully applied to the analysis of 22 internal CV samples from an internal program.

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction.

Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported. Graphical Abstract ᅟ.

Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study.

Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed. Graphical Abstract ᅟ.

Investigating changes in the gas-phase conformation of Antithrombin III upon binding of Arixtra using traveling wave ion mobility spectrometry (TWIMS).

We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII-Arixtra complex. IM measurements of ATIII and ATIII-Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII-Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein-carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.