An investigation into the correlation between visual performance in simulated complex environments and academic attainment among primary school students.

Traditional vision screenings in schools are limited to simple visual tasks, yet students in their daily learning face more complex visual environments. Binocular rivalry tasks can partially simulate the visual challenges of real visual environments and activate advanced visual processing mechanisms that simple visual tasks cannot. Therefore, by superimposing binocular rivalry-state tasks onto simple visual tasks, we have developed an innovative vision screening program to rapidly and extensively assess students' visual performance in complex environments. This is a cross-sectional study in which we investigated the performance of 1126 grade 1-6 students from a primary school in Wuxi, China, in rivalry-state stereoscopic vision tasks. The correlation between the screening results of 1044 students and their academic achievements was also statistically analyzed. The study results revealed pass rates of 53.5-60.5% across various visual tests. Specifically, for first-grade students, there was a statistically significant difference in standardized Chinese scores between the group that failed and the group that passed the rivalry-state stereoscopic vision test (- 0.49 ± 3.42 vs. 0.22 ± 0.58, t = - 2.081, P = 0.04). This result underscores the importance of focusing on the visual adaptability of first graders in complex environments.Trail registration: Ethics Committee of Affiliated Children's Hospital of Jiangnan University-Certificate number: WXCH2022-04-027.

Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins.

Recombinant adeno-associated viruses (AAVs) have emerged as the leading gene delivery platform owing to their nonpathogenic nature and long-term gene expression capability. The AAV capsid, in addition to protecting the viral genome, plays an important role in viral infectivity and gene transduction, indicating the value of the constituent viral proteins (VPs) being well-characterized as part of gene therapy development. However, the limited sample availability and sequence homology shared by the VPs pose challenges to adapt existing analytical methods developed for conventional biologics. In this study, we report the development of reversed-phase liquid chromatography/mass spectrometry-based methods for characterization of AAV capsid proteins at intact protein and peptide level with reduced sample consumptions. The developed methods allowed the measurement of VP expression with fluorescence detection and intact mass/post-translational modifications (PTMs) analysis through a benchtop time-of-flight mass spectrometer. The general applicability and validity of the methods for gene therapy product development were demonstrated by applying the optimized methods to multiple common AAV serotypes. A 1-h enzymatic digestion method was also developed using 1.25 µg of AAV VPs, providing >98% protein sequence coverage and reproducible relative quantification of various PTMs of the VPs. The efficient and sensitive analyses of AAV capsid proteins enabled by the reported methods provide further understanding and offer guidance in the development and manufacturing of AAV-related therapeutics.

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 (Tf ≥ 1.50) were observed to be reduced up to 80% to a nominal value Tf ≤ 1.2 with R.S.D. % ≤ 4%. Furthermore, recovery for two of the identified peptides exhibited increased recovery in addition to reduced peak tailing when using the column bearing the hybrid silica surface. Performance was unaffected for peaks where there were no implications of metal induced effects. Collectively, this study demonstrates that the novel hybrid silica surface can effectively reduce peak tailing for acidic residue containing peptides.

UPLC-MS assessment on the structural similarity of recombinant human erythropoietin (rhEPO) analogues from manufacturers in China for attribute monitoring.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy. Since the expiration of the patent of the innovator epoetin alfa, numerous rhEPO products have emerged in global markets. As described here, multiple complementary analytical approaches are utilized for the extensive characterization of rhEPO molecules, and more importantly for the structural comparison of the rhEPO analogues on the Chinese market. The focus of this study is placed on the overall glycosylation profiling, O-glycan profiling, and N-glycan mapping by UPLC-MS with an aim to develop an effective analytical methodology to monitor the product quality attributes of rhEPO analogues. Two rhEPO analogues manufactured in China were analyzed to demonstrate the principle of the developed methods. Each rhEPO product showed a characteristic glycoform profile with respect to the distribution of sialic acids across multi-antennary structures, the occurrence of O-glycosylation, O-acetylation on sialic acids, and the extension of N-glycan antennae with N-acetyllactosamine units. The study demonstrates that UPLC-MS is an effective analytical tool to characterize and monitor the glycosylation profiles among rhEPO analogues in order to detect and account for the divergence between rhEPO products, as well as the presence of unusual or unexpected glycans.

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. To minimize user intervention and improve assay robustness, a robotic liquid handling platform was used to automate the release and labeling of N-glycans within 2 h. To further increase the throughput, a 5 min method was developed on a liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) system using an integrated glycan library based on retention time and accurate mass. The optimized method was then applied to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. Consistent relative abundance of critical species such as high mannose and sialylated glycans was obtained for samples within the same batch (mean percent relative standard deviation [RSD] = 5.3%) with data being acquired, processed, and reported in an automated manner. The data acquisition and analysis of the 48 samples were completed within 6 h, which represents a 90% improvement in throughput compared with conventional LC-FLR-based methods. Together, this workflow facilitates the rapid screening of glycans, which can be deployed at various stages of drug development such as process optimization, bioreactor monitoring, and clone selections, where high-throughput and improved productivity are particularly desired.

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. formic acid) as mobile phase additives. Citric acid and medronic acid as metal complexing mobile phase additives were evaluated for their ability to mitigate metal-ion mediated adsorption in RPLC/MS-based peptide mapping assays. In this study, chromatographic performance was stabilized with peak tailing for peptides of interest reduced by as much as 40% in the presence of a chelator at a mobile phase concentration of 1 ppm. Performance gains were observed to be stable over a 67-hour time study with an average USP tailing factor of 1.00, % RSD = 0.64. The stabilizing effect of the chelator improved peptide mapping assay robustness with relative peak areas for target impurities calculated at 2.28% (% R.S.D. = 2.36) and 2.40% (% R.S.D. = 2.37). Collectively this study demonstrates that chelators as mobile phase additives offers a means to improve chromatographic performance for biomolecules sensitive to metal-ion mediated adsorption under formic acid-based RPLC conditions.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis.

Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate.

ASBTRACT The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.

Advanced assessment of the physicochemical characteristics of Remicade® and Inflectra® by sensitive LC/MS techniques.

In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.

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. METHODS: A UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm × 50 mm) for all separations. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. RESULTS: Trace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. CONCLUSIONS: Implementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection.

N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality.

Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics.

Characterisation of glycoproteins using a quadrupole time-of-flight mass spectrometer configured for electron transfer dissociation.

RATIONALE: Electron transfer dissociation (ETD) within ion trapping mass spectrometers has proven to be a useful tool for the characterisation of post-translational modifications. In this study, we describe the implementation of ETD upon a modified quadrupole time-of-flight (Q-ToF) system and methods for the analysis of glycoproteins. METHODS: Liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS) was performed using a hybrid quadrupole/ion mobility/oa-ToF mass spectrometer equipped with ETD functionality. 1,4-Dicyanobenzene reagent anions necessary for the ETD reaction were generated from a glow discharge region located within the ESI source block. ETD reactions occurred in the stacked ring travelling wave ion guide (located after the quadrupole mass filter and prior to the oa-ToF mass analyser). LC/ETD was performed upon 'super-charged' tryptic glycopeptide ions produced from the recombinant monoclonal antibody trastuzumab. LC/ETD was also performed on ions from the smaller glycopeptides obtained from erythropoietin. RESULTS: ETD performed upon the quadruply 'super-charged' N-linked glycopeptide ions of trastuzumab and the triply charged O-linked glycopeptide ions of erythropoietin provided both glycosylation site assignments and full sequence information, respectively. Tandem mass (MS/MS) spectra employing collision-induced dissociation (CID) were dominated by oxonium product ions hampering full peptide sequence characterisation. CONCLUSIONS: LC/ETD on the Q-ToF system proved effective at characterising a number of different N-linked glyco-forms of the tryptic peptide, EEQYNSTYR, from trastuzumab as well as glyco-forms from the O-linked tryptic peptide, EASIPPDAASAAPLR, from erythropoietin. The data demonstrates that the glycopeptide site heterogeneity of trastuzumab and erythropoietin can be accurately characterised. In addition, the post-column mixing of the super-charging reagent, m-NBA, is an effective method to increase the precursor ion charge state and to improve ETD reaction efficiency.

Accessing the reproducibility and specificity of pepsin and other aspartic proteases.

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Pepsin immobilized on high-strength hybrid particles for continuous flow online digestion at 10,000 psi.

Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles, and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen-deuterium exchange mass spectrometry (HDX MS) experiments. Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled. As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1000 psi. Prototype columns made with the BEH pepsin particles exhibited robust performance, and deuterium back-exchange was similar to that of other immobilized pepsin particles. These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.

Using hydrogen/deuterium exchange mass spectrometry to study conformational changes in granulocyte colony stimulating factor upon PEGylation.

PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring, and protein therapeutic characterization in the biopharmaceutical industry.

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry.

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 µm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.

Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MS(E) analysis of RNA oligonucleotides.

Fast and efficient ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence-related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS(E) (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS(E) technique is as efficient as the traditional MS/MS method, yet MS(E) is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2'-ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS(E) approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS(E). Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time-of-flight (QTOF) MS/MS and MS(E) methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures.

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies.

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS(E) peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.