Assessment of monoclonal antibody glycosylation: a comparative study using HRMS, NMR, and HILIC-FLD.

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.

Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Proteomics of the heart.

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein and proteoform expression, structural conformations, dynamic turnover, and protein-protein interactions. Cardiac proteomics can offer a broader and deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and it is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed researchers to study proteomes at a single-cell level and employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic posttranslational modifications. Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction, have provided the tools to study a challenging organ in the laboratory. Further technological development will pave the way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.

An assessment of AcquireX and Compound Discoverer software 3.3 for non-targeted metabolomics.

We used the Exploris 240 mass spectrometer for non-targeted metabolomics on Saccharomyces cerevisiae strain BY4741 and tested AcquireX software for increasing the number of detectable compounds and Compound Discoverer 3.3 software for identifying compounds by MS2 spectral library matching. AcquireX increased the number of potentially identifiable compounds by 50% through six iterations of MS2 acquisition. On the basis of high-scoring MS2 matches made by Compound Discoverer, there were 483 compounds putatively identified from nearly 8000 candidate spectra. Comparisons to 20 amino acid standards, however, revealed instances whereby compound matches could be incorrect despite strong scores. Situations included the candidate with the top score not being the correct compound, matching the same compound at two different chromatographic peaks, assigning the highest score to a library compound much heavier than the mass for the parent ion, and grouping MS2 isomers to a single parent ion. Because the software does not calculate false positive and false discovery rates at these multiple levels where such errors can propagate, we conclude that manual examination of findings will be required post software analysis. These results will interest scientists who may use this platform for metabolomics research in diverse disciplines including medical science, environmental science, and agriculture.

Low cost sample preparation method using ultrasound for the determination of environmentally critical elements in seaweed.

In this study, ultrasound (US) was evaluated for As, Cd, Pb, Mn, Sr and V extraction from seaweed samples. The following parameters of ultrasound-assisted extraction (UAE) using an US bath were: frequency (25 to 130 kHz), amplitude (30 to 100%), temperature (30 to 80 °C), sample mass (50 to 200 mg), extractant concentration (1 to 3 mol L-1 of HNO3) and treatment time (5 to 30 min). Acoustic density and power density distribution were calculated using the calorimetric method and mapping of the acoustic pressure distribution was also evaluated. The optimized UAE conditions were 200 mg of sample in 10 mL of 2 mol L-1 HNO3 and 30 min of sonication in a 25 kHz US bath (37.2 ± 4.0 W L-1) at 70% of amplitude and 70 °C. Analytes were quantified using inductively coupled plasma mass spectrometry and results were compared with values obtained using "silent" conditions (magnetic or mechanical stirring at 500 rpm, and without stirring), and a reference method based on microwave-assisted wet digestion (MAWD). The UAE method demonstrated the best extraction efficiency (higher than 95%) for all analytes, especially for As, Cd and V, with lower standard deviations (up to 5%) and lower blank values in comparison with the silent conditions. The proposed UAE method was more advantageous than the reference method, being faster, simpler, safer, more environmentally friendly, and with higher detectability (lower limits of quantification, from 0.0033 to 1.34 µg g-1). In addition, negligible blank values were obtained for UAE and no interference were observed in the determination step. Furthermore, the optimized UAE method was applied for Antarctic seaweed samples and comparison with results obtained by MAWD was satisfactory. In this sense, UAE is demonstrated to be a suitable option for sample preparation of seaweed samples and further determination of environmentally critical elements avoiding the use of concentrated reagents as in the MAWD reference method.

Non-destructive egg breed separation using advanced VOC analytical techniques HSSE-GC-MS, PTR-TOF-MS, and SIFT-MS: Assessment of performance and systems' complementarity.

Over the past decade, advanced analytical techniques have been utilized to examine volatile organic compounds (VOCs) in eggs. These VOCs offer valuable insights into factors such as freshness, fertility, the presence of cracks, embryo sex, and breed. In our study, we assessed three mass spectrometry-based systems (headspace sorptive extraction gas chromatography-mass spectrometry; HSSE-GC-MS, proton transfer reaction time-of-flight-mass spectrometry; PTR-TOF-MS; and selected ion flow tube mass spectrometry; SIFT-MS) to analyze and identify VOCs present in intact hatching eggs from three distinct breeds (Dekalb white layer, Shaver brown layer, and Ross 308 broiler). The eggs were sampled on incubation days 2 and 8, to identify VOCs that distinguish breeds irrespective of incubation day. VOC measurements were conducted on 15 eggs per breed by placing them together with PDMS-coated stir bars inside inert Teflon® air sampling bags. After an accumulation period of 2 h, the headspace was analyzed using PTR-TOF-MS and SIFT-MS, while the VOCs adsorbed onto the stir bars were analyzed using GC-MS for additional compound identification. Partial least squares discriminant analysis (PLS-DA) models were constructed for breed differentiation, and variable selection was performed. As a result, 111 VOCs were identified using HSSE-GC-MS, with alcohols and esters being the most abundant. The PLS-DA models demonstrated the efficacy of breed discrimination, with the HSSE-GC-MS and the PTR-TOF-MS exhibiting the highest balanced accuracy of 95.5 % using a reduced set of 11 VOCs and 5 product ions, respectively. The SIFT-MS model had a balanced accuracy of 92.8 % with a reduced set of 11 product ions. Furthermore, complementarity was observed between HSSE-GC-MS, which primarily selected higher molecular weight VOCs, and PTR-TOF-MS and SIFT-MS. A higher correlation was found for compound abundances between the HSSE-GC-MS and the PTR-TOF-MS relative to the SIFT-MS, indicating that the PTR-TOF-MS was better suited to quantify specific compounds identified by the HSSE-GC-MS. Finally, the findings support the presence of VOCs originating from both synthetic and natural sources, highlighting the ability of the VOC analysis systems to non-destructively perform quality control and reveal differences in management practices or biological information encoded in eggs.

Rapid authenticity assessment of PGI Hongyuan yak milk based on SICRIT-QTOF MS.

Hongyuan (HY) yaks live in a pollution-free environment, making HY yak milk a green food, but their short milk production period and low milk yield make yak milk precious and expensive. The phenomenon of counterfeiting HY yak milk with ordinary milk from other origins has already occurred, so the authenticity assessment of HY yak milk is necessary. This study developed a rapid soft ionisation by chemical reaction in transfer quadrupole time-of-flight mass spectrometry (SICRIT-QTOF MS) for HY yak milk differences assessment. Principal component analysis and orthogonal least squares discriminant analysis showed differences between HY milk and the other three origins. Twenty-eight differential compounds were screened out by variable importance in projection, fold change, P-value, and database matching. Furthermore, six characteristic compounds (proline, 2-hydroxy-3-methylbutyric acid, and l-isoleucine, etc.) of HY samples were putatively identified. The study demonstrated that SICRIT-QTOF MS has great potential for rapidly distinguishing the milk origin.

Using Liquid Chromatography-Isotope Ratio Mass Spectrometry for Detection of Economically Motivated Adulteration of Maple Syrup.

BACKGROUND: Maple syrup is a sought-after commodity, and used as a condiment and a sweetener. Also, it is an active target of economically motivated adulteration (EMA), similar to other foods such as lemon juice and honey. OBJECTIVE: This study is aimed to detect low cost sugar adulteration in maple syrup via an internal standard method using malic acid through solid-phase extraction (SPE) and LC with isotope ratio mass spectrometric detection (LC-IRMS). METHODS: In this work, an optimized SPE sample preparation procedure was used for the isolation of organic acids from maple syrup. Using LC-IRMS, malic acid was separated from other organic acids and the δ13C value of malic acid was determined. Eleven maple syrup samples, domestic or imported from Canada, were evaluated for 13C/12C ratios (δ13C values) using combustion module-cavity ring down spectrometry (CM-CRDS) and compared to the δ13C values obtained from well-established elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) methods. The δ13C values of isolated malic acid analyzed by SPE-LC-IRMS were used as internal standards and compared to the δ13C values of bulk maple syrup; difference (δ13Csugars - δ13Cmalic acid) values greater than 3.6‰ are indicative of low-cost sugar adulteration. RESULTS: Overall, the results obtained from SPE-LC-IRMS provided a faster, novel analysis approach for determining low-cost sugar adulteration in maple syrup for regulatory purposes. This method also provided lower detectable limits of adulteration versus current literature reports using bulk analysis and comparable detection limits to Tremblay and co-workers who utilized an internal standard method. CONCLUSION: SPE-LC-IRMS is a robust method that can be used for detecting adulteration in maple syrup samples for regulatory purposes. HIGHLIGHTS: SPE-LC-IRMS is a faster, novel analysis approach for determining C4 adulteration in maple syrup with lower detection limits.

Applying a risk assessment guided evaluation for verifying comprehensive two-dimensional gas chromatography to analyse complex pharmaceuticals.

The reliability of analytical results is critical and indispensable when applied in regulated environments such as the pharmaceutical industry. Therefore, analytical workflows must be validated. However, validation guidelines are often designed for quantitative targeted analysis and rarely apply to qualitative untargeted approaches. In this study, we employ a risk assessment approach to identify critical parameters which might influence the qualitative results derived by online derivatisation - comprehensive two-dimensional gas chromatography coupled to a high-resolution time-of-flight mass spectrometer (GC × GC-HR-ToF-MS) for the analysis of the active pharmaceutical ingredient (API) sodium bituminosulfonate (SBS). To show the complexity and feasibility of such an approach, we focus on investigating three potential risk factors: sample preparation, vapourability, and the thermal stability of sulfonates. Through the individual evaluation of these potential risk factors due to the application of sample preparation approaches and thermal gravimetric analysis (TGA), we demonstrate the high derivatisation efficiency and repeatability of the online derivatisation method and confirm the absence of derivatisation-induced side reactions. In addition, we also show the potential thermal instability of an incompletely derivatised API. To address the limitation of these individual assessments, we applied a holistic evaluation step with negative electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI( -) FT-ICR MS) as an orthogonal technique. This confirms that most of the API is detected via the presented GC-based method. Thereby, we demonstrated the practical feasibility of the risk assessment-based approach to ensure the validity of the qualitative data for a complex untargeted method.

Structural Elucidation of Agrochemical Metabolic Transformation Products Based on Infrared Ion Spectroscopy to Improve In Silico Toxicity Assessment.

Toxicological assessments of newly developed agrochemical agents consider chemical modifications and their metabolic and biotransformation products. To carry out an in silico hazard assessment, understanding the type of chemical modification and its location on the original compound can greatly enhance the reliability of the evaluation. Here, we present and apply a method based on liquid chromatography-mass spectrometry (LC-MS) enhanced with infrared ion spectroscopy (IRIS) to better delineate the molecular structures of transformation products before in silico toxicology evaluation. IRIS facilitates the recording of IR spectra directly in the mass spectrometer for features selected by retention time and mass-to-charge ratio. By utilizing quantum-chemically predicted IR spectra for candidate molecular structures, one can either derive the actual structure or significantly reduce the number of (isomeric) candidate structures. This approach can assist in making informed decisions. We apply this method to a plant growth stimulant, digeraniol sinapoyl malate (DGSM), that is currently under development. Incubation of the compound in Caco-2 and HepaRG cell lines in multiwell plates and analysis by LC-MS reveals oxidation, glucuronidation, and sulfonation metabolic products, whose structures were elucidated by IRIS and used as input for an in silico toxicology assessment. The toxicity of isomeric metabolites predicted by in silico tools was also assessed, which revealed that assigning the right metabolite structure is an important step in the overall toxicity assessment of the agrochemical. We believe this identification approach can be advantageous when specific isomers are significantly more hazardous than others and can help better understand metabolic pathways.

Internal Fragment Ions from Higher Energy Collision Dissociation Enable the Glycoform-Resolved Asn325 Deamidation Assessment of Antibodies by Middle-Down Mass Spectrometry.

A major challenge in proteoform characterization is to obtain information on coexisting post-translational modifications (PTMs), which is lost in traditional bottom-up analysis. Middle-down approaches of antibodies provide a good balance of resolution, site-specificity, and proteoform heterogeneity to characterize individual proteoforms at subunit level. Currently, most middle-down studies focus on terminal fragment ions, which may not cover or resolve PTMs in the center of the sequence or with minor mass shifts such as deamidation, often a critical quality attribute for antibody drugs. Antibody glycosylation at Asn 297 and deamidation at Asn 325 are two important PTMs impacting the interaction with Fc gamma receptors and hence effector functions such as antibody-dependent cellular cytotoxicity. Here, we established a new middle-down workflow that uses internal fragment ions for the qualitative and quantitative assessment of a functional relevant deamidation site, Asn 325, through higher energy collision dissociation fragmentation of individual antibody glycoforms upon quadrupole isolation. We identified a signature internal fragment ion to resolve and estimate the relative abundances of deamidation of individual glycoforms in complex mixtures. Our proof-of-concept work demonstrates the feasibility to identify and quantify Asn 325 deamidation at the glycoform-resolved subunit level using internal fragment ions, which greatly advances the capabilities to study PTM dynamics by middle-down analysis.

Longitudinal Plant Health Monitoring via High-Resolution Mass Spectrometry Screening Workflows: Application to a Fertilizer Mediated Tomato Growth Experiment.

Significant efforts have been spent in the modern era towards implementing environmentally friendly procedures like composting to mitigate the negative effects of intensive agricultural practices. In this context, a novel fertilizer was produced via the hydrolysis of an onion-derived compost, and has been previously comprehensively chemically characterized. In order to characterize its efficacy, the product was applied to tomato plants at five time points to monitor plant health and growth. Control samples were also used at each time point to eliminate confounding parameters due to the plant's normal growth process. After harvesting, the plant leaves were extracted using aq. MeOH (70:30, v/v) and analyzed via UPLC-QToF-MS, using a C18 column in both ionization modes (±ESI). The data-independent (DIA/bbCID) acquisition mode was employed, and the data were analyzed by MS-DIAL. Statistical analysis, including multivariate and trend analysis for longitudinal monitoring, were employed to highlight the differentiated features among the controls and treated plants as well as the time-point sequence. Metabolites related to plant growth belonging to several chemical classes were identified, proving the efficacy of the fertilizer product. Furthermore, the efficiency of the analytical and statistical workflows utilized was demonstrated.

Recent advances in structural mass spectrometry methods in the context of biosimilarity assessment: from sequence heterogeneities to higher order structures.

Biotherapeutics and their biosimilar versions have been flourishing in the biopharmaceutical market for several years. Structural and functional characterization is needed to achieve analytical biosimilarity through the assessment of critical quality attributes as required by regulatory authorities. The role of analytical strategies, particularly mass spectrometry-based methods, is pivotal to gathering valuable information for the in-depth characterization of biotherapeutics and biosimilarity assessment. Structural mass spectrometry methods (native MS, HDX-MS, top-down MS, etc.) provide information ranging from primary sequence assessment to higher order structure evaluation. This review focuses on recent developments and applications in structural mass spectrometry for biotherapeutic and biosimilar characterization.

Untargeted metabolomics liquid chromatography-high resolution mass spectrometry approach for the geographical origin assessment of Italian dehydrated apples.

Geographical provenience is nowadays a relevant aspect of the authenticity and the quality of many food commodities. Dehydrated apple cubes/slices represent an ingredient commonly used by food companies for bakery products. However, this apple-based matrix is not so known and studied from an analytical point of view. In the present work, seven compounds were identified as key molecules to distinguish between Italian and non-Italian samples, through an untargeted ultrahigh-pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) approach. This methodology was merged with multivariate statistical analysis, and the principal features were studied and identified considering several identification steps. Samples from 2020 and 2021 harvesting campaigns, with partial and total dehydration rates, with or without peel, and from different apple varieties were considered for the study, for a total of 91 samples. Afterward, the same analysis protocol was applied to an external set (n = 12 samples), included in the statistical models, searching for the key compounds identified in the training set. Interesting and significant results underlined the potentiality of the UHPLC-HRMS technology as a confirmatory strategy for the geographical origin assessment of dehydrated apple commodities.

Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Equivalence assessment of biotherapeutics with N- and O-glycosylation sites by sequential intact glycoform mass spectrometry (IGMS).

Glycosylation is a crucial attribute for biotherapeutics with significant impacts on quality, stability, safety, immunogenicity, pharmacokinetics, and efficacy. Therefore, to ensure consistent glycosylation, a systematic review of biotherapeutics is absolutely required including the variable glycan structure (micro-heterogeneity) and different occupancy at individual site (macro-heterogeneity) from drug design to upstream and downstream bioprocesses. Various methods have been used for glyco-characterization of biotherapeutics at the glycan, glycopeptide, and intact protein levels. In particular, intact protein analysis is considered a facile and rapid glycoform monitoring approach used throughout the product development lifecycle to determine suitable glycosylation lead candidates and reproducible product quality. However, intact glycoform characterization of diverse and complex biotherapeutics with multiple N- and O-glycosylation sites can be very challenging. To address this, a robust analytical platform that enables rapid and accurate characterization of a biotherapeutics with highly complex multiple glycosylation using two-step intact glycoform mass spectrometry has been developed. We used darbepoetin alfa, a second-generation EPO bearing multiple N- and O-glycosylation sites, as a model biotherapeutics to obtain integrated information on glycan heterogeneity and site occupancy through step-by-step MS of intact protein and enzyme-treated protein. In addition, we performed a comparative assessment of the heterogeneity from different products, confirming that our new method can efficiently evaluate glycosylation equivalence. This new strategy provides rapid and accurate information on the degree of glycosylation of a therapeutic glycoprotein with multiple glycosylation, which can be used to assess glycosylation similarity between batches and between biosimilar and reference during development and production.

Cation exchange chromatography on a monodisperse 3 µm particle enables extensive analytical similarity assessment of biosimilars.

Biosimilarity assessment requires extensive characterization and comparability exercises to investigate product quality attributes of an originator product and its potential biosimilar(s) and to highlight any differences between them. Performing a thorough comparison allows a shortened approval path, which also eliminates lengthy and expensive clinical trials, ensuring comparable product quality and efficacy but at lower drug prices. The wide variety of analytical methods available for biosimilar assessment ranges from biological to analytical assays, each providing orthogonal information to fully characterize biosimilar candidates. Intact native mass spectrometry (MS) has been shown to be an excellent tool for detection and monitoring of important quality attributes such as N-glycosylation, deamidation, sequence truncation and higher order structures. When combined with efficient upfront separation methods, simplification of the proteoform heterogeneity and associated complexity prior to MS analysis can be achieved. Native mass spectrometry can provide robust and accurate results within short analysis times and requires minimal sample preparation. In this study we report the use of a monodisperse strong cation exchange chromatography phase hyphenated with Orbitrap mass spectrometry (SCX-MS) to compare the best-selling biopharmaceutical product Humira® with 7 commercially approved biosimilar products. SCX-MS analysis allowed for the identification of previously described as well as so far unreported proteoforms and their relative quantitation across all samples, revealing differences in N-glycosylation and lysine truncation, as well as unique features for some products such as sialylation and N-terminal clipping. SCX-MS analysis, powered by a highly efficient separation column, enabled deep and efficient analytical comparison of biosimilar products.

Assessment of a 60-Biomarker Health Surveillance Panel (HSP) on Whole Blood from Remote Sampling Devices by Targeted LC/MRM-MS and Discovery DIA-MS Analysis.

Telehealth, accessing healthcare and wellness remotely, should be a cost-effective and efficient way for individuals to receive care. The convenience of having a reliable remote collection device for blood tests will facilitate access to precision medicine and healthcare. Herein, we tested a 60-biomarker health surveillance panel (HSP), containing 35 FDA/LDT assays and covering at least 14 pathological states, on 8 healthy individuals' ability to collect their own capillary blood from a lancet finger prick and directly compared it to the traditional phlebotomist venous blood and plasma collection methods. All samples were spiked with 114 stable-isotope-labeled (SIL) HSP peptides and quantitatively analyzed by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM-MS) scheduled method targeting 466 transitions from 114 HSP peptides and by a discovery data-independent acquisition mass spectrometry (DIA-MS) method. The average peak area ratio (PAR) of the HSP quantifier peptide transitions from all 8 volunteers' capillary blood (n = 48), venous blood (n = 48), and matched plasma (n = 24) was <20% coefficients of variation (CV). Heat map analysis of all 8 volunteers demonstrated that each individual had a unique biosignature. Biological replicates from capillary blood and venous blood clustered within each volunteer in k-means clustering analysis. Pearson statistical analysis of the three biofluids indicated that there was >90% similarity. Discovery DIA-MS analysis of the same samples using a plasma spectral library and a pan-human spectral library identified 1121 and 4661 total proteins, respectively. In addition, at least 122 FDA-approved biomarkers were identified. DIA-MS analysis reproducibly quantitated (<30% CV) ∼600-700 proteins in capillary blood, ∼800 proteins in venous blood, and ∼300-400 proteins in plasma, demonstrating that an expansive biomarker panel is possible with current mass spectrometry technology. Both targeted LC/MRM-MS and discovery DIA-MS analysis of whole blood collected on remote sampling devices are viable options for personal proteome biosignature stratification in precision medicine and precision health.

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Technological development of multidimensional liquid chromatography-mass spectrometry in proteome research.

Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for high-throughput proteomic research. Limited by the peak capacity, the separation performance of conventional single-dimensional LC hampers the development of proteomics. Combining different separation modes orthogonally, multidimensional liquid chromatography (MDLC) with high peak capacity was developed to address this challenge. MDLC has evolved rapidly since its establishment, and the progress of proteomics has been greatly facilitated by the advent of novel MDLC-MS-based methods. In this paper, we will review the advances of MDLC-MS-based methodologies and technologies in proteomics studies, from different perspectives including novel application scenarios and proteomic targets, automation, miniaturization, and the improvement of the classic methods in recent years. In addition, attempts regarding new MDLC-MS models are also mentioned together with the outlook of MDLC-MS-based proteomics methods.