RESUMEN

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

Asunto(s)

RESUMEN

Many methods using liquid chromatography-mass spectrometry (LC-MS) have been established for identifying residual host cell proteins (HCPs) to aid in the process development and quality control of therapeutic proteins. However, the use of MS-based techniques for adeno-associated virus (AAV) is still in its infancy, with few methods reported and minimal information available on potentially problematic HCPs. In this study, we developed a highly sensitive and effective differential digestion method to profile residual HCPs in AAV. Unlike direct digestion, which completely digests both AAV and HCPs, our differential digestion method takes advantage of AAV's unique characteristics to maintain the integrity of AAV while preferentially digesting HCPs under denaturing and reducing conditions. This differential digestion method requires only several micrograms of sample and significantly enhances the identification of HCPs. Furthermore, this method can be applied to all five different AAV serotypes for comprehensive HCP profiling. Our work fills a gap in AAV HCP analysis by providing a sensitive and robust strategy for detecting, monitoring, and measuring HCPs.

Asunto(s)

RESUMEN

Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying all problematic HCPs present at low levels remain challenging. Here, we developed a practical, effective strategy for the identification and quantification of low abundance HCPs, which facilitates the improvement of downstream purification process to eliminate potentially problematic HCPs. A case study of using this strategy to investigate a problematic HCP is presented. Initially, a commonly used native digestion approach coupled with UPLC-MS/MS was applied for HCP profiling, wherein several lipases and proteases were identified in a monoclonal antibody named mAb1 in early stages of purification process development. A highly active lipase, liver carboxylesterase (CES), was found to be responsible for polysorbate 80 degradation. To facilitate process improvement, after the identification of CES, we developed a highly sensitive LC-MS/MS-MRM assay with a lower limit of quantification of 0.05 ppm for routine monitoring of the CES in mAb1 produced through the different processes. This workflow was applied in low-level lipase identification and absolute quantification, which facilitated the investigation of polysorbate degradation and downstream purification improvement to further remove the problematic HCP. The current MRM method increased the sensitivity of HCP quantification by over 10-fold that in previously published studies, thus meeting the needs for quantification of problematic HCPs at sub-ppm to ppb levels during drug development. This workflow could be readily adapted to the detection and quantification of other problematic HCPs present at extremely low levels in therapeutic protein drug candidates.

Asunto(s)

RESUMEN

High molecular weight (HMW) species are product-related variants that may impact therapeutic product safety and efficacy. Therefore, HMW species and aggregates are considered critical quality attributes and their levels should be closely monitored and controlled during drug development, commercial manufacturing, and shelf-life storage period for therapeutic monoclonal antibody drug products. Various biophysical and analytical methods have been developed to characterize the HMW species to understand their mechanisms of formation and assess potential product risk. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs are identified in the enriched HMW than in the DS, thus demonstrating a potential interaction between HCPs and HMW. Certain HCPs, including commonly detected HCPs and problematic HCPs, were enriched in HMW fractions. Especially, the most abundant HCP from mAb1, CC motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The CC motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the CC motif chemokine level in the final mAb1 DS. Our findings demonstrate that some HCPs are more preferentially bound to HMW species and this finding may provide a new opportunity for removing HCPs in downstream purification steps.

Asunto(s)

RESUMEN

Background: There is still no consensus regarding the role of laparoscopy in trauma cases. The purpose of this paper is to assess the value of diagnostic and therapeutic laparoscopy for patients with blunt or penetrating abdominal trauma by performing a systematic review and meta-analysis. Methods: PubMed, Embase, and the Cochrane library were systemically searched for the randomized controlled trials (RCTs) and non-RCT comparative studies on effectiveness and safety of laparoscopy vs. laparotomy for the two authors independently performed the search, data extraction, and quality assessment. Results: A total of 5,517 patients were enrolled in 23 eligible studies that were published in English. Meta-analysis results suggest that there is no significant difference in the incidence of missed injury and mortality between abdominal trauma patients receiving laparoscopy and those receiving laparotomy. Concerning postoperative complications, compared with patients in the open surgery group, those in the laparoscopy group are at a similar risk of intra-abdominal abscesses, thromboembolism, and ileus, while there is a decreased incidence of wound infection and pneumonia. Besides, patients in the laparoscopy group experience shorter hospitalization times and procedure times. For most outcomes, the sensitivity analysis yielded similar results to the primary analysis. Conclusion: Laparoscopic surgery is a practical alternative to laparotomy for appropriate patients. The decision to perform laparoscopy should be based on the experience of the surgeon and the resources available.

RESUMEN

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Asunto(s)

RESUMEN

An effective control strategy is critical to ensure the safety, purity and potency of biopharmaceuticals. Appropriate analytical tools are needed to realize such goals by providing information on product quality at an early stage to help understanding and control of the manufacturing process. In this work, a fully automated, multi-capillary instrument is utilized for size-based separation and western blot analysis to provide an early readout on product quality in order to enable a more consistent manufacturing process. This approach aims at measuring two important qualities of a biopharmaceutical protein, titer and isoform distribution, in cell culture harvest samples. The acquired data for isoform distribution can then be used to predict the corresponding values of the final drug substance, and potentially provide information for remedy through timely adjustment of the downstream purification process, should the expected values fall out of the accepted range.

Asunto(s)

RESUMEN

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

Asunto(s)

RESUMEN

LC-MS/MS is one of the most powerful tools for N-glycan structure elucidation; however, it is still challenging to identify some glycan structures with low abundance. In this study, we investigated the chromatographic behavior of permethylated N-glycans. The relationship between retention times versus molecular weight of dextran, dextrin, and model glycans was investigated. Also, the nonpolar surface area of glycans was calculated and compared to their experimental retention times. Both retention time and nonpolar surface area trends are similar when the intermolecular interaction is included in the calculation. Moreover, retention time corresponds to glycan types and branch types. The N-glycans analysis model, which combines high mass accuracy and retention time, was applied to confirm serum N-glycans. In total, there were 78 N-glycan compositions identified. A linear relationship between retention times and molecular weights were observed for each subgroup of glycan structures, for example, R(2) value for complex N-glycans was determined to be > 0.98. Moreover, the retention time could be further applied to distinguish between structural isomers as well as linkage isomers. MS/MS data were used to confirm the structural isomers.

Asunto(s)

RESUMEN

Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification.

Asunto(s)

RESUMEN

Prostate specific antigen (PSA) is currently used as a diagnostic biomarker for prostate cancer. It is a glycoprotein possessing a single glycosylation site at N69. During our previous study of PSA N69 glycosylation, additional glycopeptides were observed in the PSA sample that were not previously reported and did not match glycopeptides of impure glycoproteins existing in the sample. This extra glycosylation site of PSA is associated with a mutation in KLK3 genes. Among single nucleotide polymorphisms (SNPs) of KLKs families, the rs61752561 in KLK3 genes is an unusual missense mutation resulting in the conversion of D102 to N in PSA amino acid sequence. Accordingly, a new N-linked glycosylation site is created with an N102MS motif. Here we report the first qualitative and quantitative glycoproteomic study of PSA N102 glycosylation site by LC-MS/MS. We successfully applied tandem MS to verify the amino acid sequence possessing N102 glycosylation site and associated glycoforms of PSA samples acquired from different suppliers. Among the three PSA samples, HexNAc2Hex5 was the predominant glycoform at N102, while HexNAc4Hex5Fuc1NeuAc1 or HexNAc4Hex5Fuc1NeuAc2 was the primary glycoforms at N69. D102 is the first amino acid of "kallikrein loop", which is close to a zinc-binding site and catalytic triad. The different glycosylation of N102 relative to N69 might be influenced by the close vicinity of N102 to these functional sites and steric hindrance.

Asunto(s)

RESUMEN

Although MYCN amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, MYCN-nonamplified neuroblastoma from high-risk, MYCN-amplified disease are largely unknown. Genomic and proteomic studies have been limited in discerning differences in signaling pathways that account for this heterogeneity. N-Linked glycosylation is a common protein modification resulting from the attachment of sugars to protein residues and is important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proven to be useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between MYCN-nonamplified SY5Y and MYCN-amplified NLF cell lines with the aim of identifying changes in sugar abundance linked to high-risk neuroblastoma. Through a combination of liquid chromatography-mass spectrometry and bioinformatics analysis, we identified 16 glycans that show a statistically significant change in abundance between NLF and SY5Y samples. Closer examination revealed the preference for larger (in terms of total monosaccharide count) and more sialylated glycan structures in the MYCN-amplified samples in comparison to smaller, nonsialylated glycans that are more dominant in the MYCN-nonamplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis.

Asunto(s)

RESUMEN

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 µL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a µL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Asunto(s)

RESUMEN

RATIONALE: Liquid chromatography/mass spectrometry (LC/MS) is currently considered to be a conventional glycomics analysis strategy due to the high sensitivity and ability to handle complex biological samples. Interpretation of LC/MS data is a major bottleneck in high-throughput glycomics LC/MS-based analysis. The complexity of LC/MS data associated with biological samples prompts the needs to develop computational tools capable of facilitating automated data annotation and quantitation. METHODS: An LC/MS-based automated data annotation and quantitation software, MultiGlycan-ESI, was developed and utilized for glycan quantitation. Data generated by the software from LC/MS analysis of permethylated N-glycans derived from fetuin were initially validated by manual integration to assess the performance of the software. The performance of MultiGlycan-ESI was then assessed for the quantitation of permethylated fetuin N-glycans analyzed at different concentrations or spiked with permethylated N-glycans derived from human blood serum. RESULTS: The relative abundance differences between data generated by the software and those generated by manual integration were less than 5%, indicating the reliability of MultiGlycan-ESI in quantitation of permethylated glycans analyzed by LC/MS. Automated quantitation resulted in a linear relationship for all six N-glycans derived from 50 ng to 400 ng fetuin with correlation coefficients (R(2) ) greater than 0.93. Spiking of permethylated fetuin N-glycans at different concentrations in permethylated N-glycan samples derived from a 0.02 µL of HBS also exhibited linear agreement with R(2) values greater than 0.9. CONCLUSIONS: With a variety of options, including mass accuracy, merged adducts, and filtering criteria, MultiGlycan-ESI allows automated annotation and quantitation of LC/ESI-MS N-glycan data. The software allows the reliable quantitation of glycan LC/MS data. The software is reliable for automated glycan quantitation, thus facilitating rapid and reliable high-throughput glycomics studies.

Asunto(s)

RESUMEN

Defining clinically relevant biomarkers for early stage hepatocellular carcinoma (HCC) in a high-risk population of cirrhotic patients has potentially far-reaching implications for disease management and patient health. Changes in glycan levels have been associated with the onset of numerous diseases including cancer. In the present study, we used liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) to analyze N-glycans in sera from 183 participants recruited in Egypt and the U.S. and identified candidate biomarkers that distinguish HCC cases from cirrhotic controls. N-Glycans were released from serum proteins and permethylated prior to the LC-ESI-MS analysis. Through two complementary LC-ESI-MS quantitation approaches, global profiling and targeted quantitation, we identified 11 N-glycans with statistically significant differences between HCC cases and cirrhotic controls. These glycans can further be categorized into four structurally related clusters, matching closely with the implications of important glycosyltransferases in cancer progression and metastasis. The results of this study illustrate the power of the integrative approach combining complementary LC-ESI-MS based quantitation approaches to investigate changes in N-glycan levels between HCC cases and patients with liver cirrhosis.

Asunto(s)

RESUMEN

UNLABELLED: As a common post-translational modification, protein glycosylation plays an important role in many biological processes, and it is known to be associated with human diseases. Mass spectrometry (MS)-based glycomic profiling techniques have been developed to measure the abundances of glycans in complex biological samples and applied to the discovery of putative glycan biomarkers. To automate the annotation of glycomic profiles in the liquid chromatography-MS (LC-MS) data, we present here a user-friendly software tool, MultiGlycan, implemented in C# on Windows systems. We tested MultiGlycan by using several glycomic profiling datasets acquired using LC-MS under different preparations and show that MultiGlycan executes fast and generates robust and reliable results. AVAILABILITY: MultiGlycan can be freely downloaded at http://darwin.informatics.indiana.edu/MultiGlycan/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Asunto(s)

RESUMEN

RATIONALE: Mass spectrometry based comparative glycomics is essential for disease biomarker discovery. However, developing a reliable quantification method is still a challenging task. METHODS: We here report an isotopic labeling strategy employing stable isotopic iodomethane for comparative glycomic profiling by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). N-Glycans released from model glycoproteins and blood serum samples were permethylated with iodomethane ('light') and iodomethane-d1 or -d3 ('heavy') reagents. Permethylated samples were then mixed at equal volumes prior to LC/ESI-MS analysis. RESULTS: Peak intensity ratios of N-glycans isotopically permethylated (Heavy/Light, H/L) were almost equal to the theoretical values. Observed differences were mainly related to the purity of 'heavy' iodomethane reagents (iodomethane-d1 or -d3). The data suggested the efficacy of this strategy to simultaneously quantify N-glycans derived from biological samples representing different cohorts. Accordingly, this strategy is effective in comparing multiple samples in a single LC/ESI-MS analysis. The potential of this strategy for defining glycomic differences in blood serum samples representing different esophageal diseases was explored. CONCLUSIONS: LC/ESI-MS comparative glycomic profiling of isotopically permethylated N-glycans derived from biological samples and glycoproteins reliably defined glycan changes associated with biological conditions or glycoproteins expression. As a biological application, this strategy permitted the reliable quantification of glycomic changes associated with different esophageal diseases, including high grade dysplasia, Barrett's disease, and esophageal adenocarcinoma.

Asunto(s)

RESUMEN

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-µL aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.

Asunto(s)

RESUMEN

The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed.

Asunto(s)

RESUMEN

For decades, the association between aberrant glycosylation and many types of cancers has been shown. However, defining the changes of glycan structures has not been demonstrated until recently. This has been facilitated by the major advances in MS and separation science, which allows the detailed characterization of glycan changes associated with cancer. MS glycomics methods have been successfully employed to compare the glycomic profiles of different human specimens collected from disease-free individuals and patients with cancer. Additionally, comparing the glycomic profiles of glycoproteins purified from specimen collected from disease-free individuals and patients with cancer has also been performed. These types of glycan analyses employing MS or LC-MS allow the characterization of native, labeled and permethylated glycans. This review discusses the different glycomic and glycoproteomic methods employed for defining glycans as cancer biomarkers of different organs, including breast, colon, esophagus, liver, lung, ovarian, pancreas and prostate.

Asunto(s)