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The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.

Mass spectrometry‐based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom‐up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal—in primary structure—concurrent post‐translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom‐up proteomics report the average PTM status and higher‐order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low‐copy number proteins below signal‐to‐noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top‐down proteomics as an alternative. [ABSTRACT FROM AUTHOR]

Amorphous polymer/crystalline polymer blends can be prepared via the simultaneous polymerization of polymethacrylate/polyurethane combinations. The relationship between higher order structures and fracture mechanisms in these blends must be uncovered to elucidate the source of the increased fracture toughness of such materials. The present work involves the production of blended polymethacrylate/polyurethane and assess the internal structures of these specimens using optical and electron microscopy. These observations reveal the presence of both spherulites and elastomeric phases. The spherulites consisting of the polyurethane and are several micrometers in diameter whereas the phase‐separated polyurethane elastomeric domains are approximately 100 nm in size. Multiple cracks, crack bridging and plastic deformation around the precrack tips of loaded specimens are evidently responsible for the increased toughness of these blends. The former two phenomena are attributed to the presence of spherulites while the plastic deformation of the methacrylate matrix is ascribed to cavitation of the polyurethane elastomeric phases in response to loading. [ABSTRACT FROM AUTHOR]

Context. Concerning the epithelial tissue in the skin, including hair, vitamin A is required for the integrity of epithelial cells, strengthened by compounding keratin fibres. Further, the complex hormonal changes seen in pregnancy may contribute to the hair growth. Thus, the structure of the cattle coat hair may change depending on the conditions of vitamin A deficiency or pregnancy. Aims. The effects of several diseases, i.e. vitamin A deficiency, pneumonia, chronic pneumonia, and chronic renal failure, or pregnancy, on the structure of the coat hair were investigated. Methods. Physical properties of the coat hair, collected from Japanese black cattle (Bos Taurus) and Holstein cows (Bos taurus Taurus), were investigated to study the effects of vitamin A deficiency, other diseases, or pregnancy on the formation of hair. Key results. When vitamin A concentration was lowered to about half of the minimum concentration for vitamin A adequacy of 20 µg/dL in plasma (i.e. deficiency level was high), we could perceive the interference in the self-assembly of the keratin fibrils. However, we also confirmed structural modifications of the keratin fibres obtained from the cattle suffering chronic renal failure. The coat hair collected from the pregnant cows tended to show superior tensile strengths to those of the hair from the non-pregnant ones when compared at the same diameter level, although pregnancy might have increased cortisol affecting the hair follicle function. Further, from the X-ray diffraction measurements, the non-pregnant cow coat hairs, having exhibited the inferior tensile properties, were found to be structurally modified. Conclusions. The structure of the coat hair could be modified when the vitamin A concentration was severely reduced (vitamin A deficiency). The physiological conditions during pregnancy may be advantageous to serve the regular formation of the hair. Implications. Hair has a function serving as a proxy of health status for a certain period of animal life. [ABSTRACT FROM AUTHOR]

ABSTRACTThe clinical efficacy and safety of protein-based drugs such as monoclonal antibodies (mAbs) rely on the integrity of the protein higher order structure (HOS) during product development, manufacturing, storage, and patient administration. As mAb-based drugs are becoming more prevalent in the treatment of many illnesses, the need to establish metrics for quality attributes of mAb therapeutics through high-resolution techniques is also becoming evident. To this end, here we used a forced degradation method, time-dependent oxidation by hydrogen peroxide, on the model biotherapeutic NISTmAb and evaluated the effects on HOS with orthogonal analytical methods and a functional assay. To monitor the oxidation process, the experimental workflow involved incubation of NISTmAb with hydrogen peroxide in a benchtop nuclear magnetic resonance spectrometer (NMR) that followed the reaction kinetics, in real-time through the water proton transverse relaxation rate R2(1H2O). Aliquots taken at defined time points were further analyzed by high-field 2D 1H-13C methyl correlation fingerprint spectra in parallel with other analytical techniques, including thermal unfolding, size-exclusion chromatography, and surface plasmon resonance, to assess changes in stability, heterogeneity, and binding affinities. The complementary measurement outputs from the different techniques demonstrate the utility of combining NMR with other analytical tools to monitor oxidation kinetics and extract the resulting structural changes in mAbs that are functionally relevant, allowing rigorous assessment of HOS attributes relevant to the efficacy and safety of mAb-based drug products.

As microRNAs (miRNAs) are involved in many essential biological processes, their abnormal expressions can serve as biomarkers and prognostic indicators to prevent the development of complex diseases, thus providing accurate early detection and prognostic evaluation. Although a number of computational methods have been proposed to predict miRNA–disease associations (MDAs) for further experimental verification, their performance is limited primarily by the inadequacy of exploiting lower order patterns characterizing known MDAs to identify missing ones from MDA networks. Hence, in this work, we present a novel prediction model, namely HiSCMDA, by incorporating higher order network structures for improved performance of MDA prediction. To this end, HiSCMDA first integrates miRNA similarity network, disease similarity network and MDA network to preserve the advantages of all these networks. After that, it identifies overlapping functional modules from the integrated network by predefining several higher order connectivity patterns of interest. Last, a path-based scoring function is designed to infer potential MDAs based on network paths across related functional modules. HiSCMDA yields the best performance across all datasets and evaluation metrics in the cross-validation and independent validation experiments. Furthermore, in the case studies, 49 and 50 out of the top 50 miRNAs, respectively, predicted for colon neoplasms and lung neoplasms have been validated by well-established databases. Experimental results show that rich higher order organizational structures exposed in the MDA network gain new insight into the MDA prediction based on higher order connectivity patterns. [ABSTRACT FROM AUTHOR]

Graph representation learning has been extensively studied in recent years. It has been proven effective in network analysis and mining tasks such as node classification and link prediction. Learning method based on neural network has become mainstream solution because it can efficiently preserve nonlinear characteristics of graph. However, more efficient ways to encode complex graph structures are still being explored. In this paper, for undirected graphs, we present a novel Motif-Aware Generative Adversarial Network (MotifGAN) model to learn graph representation based on a re-weighted graph, which unifies both the Motif higher order structure and the original lower order structure. Firstly, we obtain the motif adjacency matrix by motif mining, which encodes the Motif higher order structure. Then we couple the motif adjacency matrix with the adjacency matrix of the original graph to get a reweighted matrix named motif connectivity matrix. The motif connectivity distribution implicit in the motif connectivity matrix is the target structure information we need to preserve. How to preserve a wealth of structural information is a challenge. Inspired by related applications of GAN, we formulate a GAN model to generate embedding representations that satisfy the target structure conditions. The GAN model usually consists of two components: generator and discriminator. Our generator tries to approximate the motif connectivity distribution, while the discriminator detects whether the connectivity of two vertexes is from ground truth or generated by the generator. In adversarial training, both parts can alternately and iteratively boost their performance. Experimental results on public datasets show that MotifGAN has made substantial progress in various applications, including link prediction, node classification and visualization.

This article is the content of the closing lecture delivered at the XIVth Manuel Rico Advanced NMR Course, held in Jaca (Spain) in June 2022. It is my personal impression on the technical frontiers where I expect new advances to expand even further the fields of NMR application, especially in the fields of biomedicine and biotechnology. It is dedicated to Robert Konrat on the occasion of his 60th birthday as an acknowledgement of his wide range of interest and vast scientific culture.

Monoclonal antibody (mAb) aggregation can present major challenges for the development of biotherapeutics. An understanding of the molecular mechanisms of mAb aggregation is highly desirable both because it allows the performance of informed risk assessments regarding the criticality of mAb aggregates and because it may facilitate rational stabilization of aggregation prone regions. Here, we report the generation and isolation of dimer species of an IgG4 mAb (mAb1) that were present in stressed material under differing levels of temperature stress. We demonstrate the power of combining established higher order techniques with non-routine analysis, such as small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein conformational array enzyme-linked immunosorbent assay (PCA ELISA), and show that dimer species formed under temperature stress are structurally distinct from those present in unstressed mAb1. Specifically, stress-induced dimers are shown to adopt a more elongated conformation with a greater degree of unfolding when compared to native dimers. Analysis by HDX-MS and PCA ELISA, supported by in silico shape and charge molecular docking, enabled the identification of residues in both the variable and constant domains that appear to play a significant role in the dimerization of mAb1. Furthermore, we show that dimers formed under temperature stress are significantly more long-lived than those present in unstressed mAb1. We also present evidence that mAb1 dimers can behave as aggregation nuclei, and that dimers produced under high-temperature stress do so to a greater extent. This work presents an advancement in our understanding of the molecular mechanisms of mAb aggregation and highlights the importance of structural characterization of dimer species during the development of mAb biotherapeutics.Abbreviations: 2DSA: 2-Dimensional Spectrum Analysis; CD: Circular Dichroism; CDR: Complementarity-Determining Region; CQA: Critical Quality Attribute; DSC: Differential Scanning Calorimetry; FTIR: Fourier Transform Infrared spectroscopy; HDX-MS: Hydrogen/Deuterium Exchange Mass Spectrometry; HIC: Hydrophobic interaction chromatography; HMWS: High Molecular Weight Species; HOS: Higher Order Structure; mAb: Monoclonal Antibody; MD: Molecular Dynamics PCA; ELISA: Protein Conformational Array Enzyme-Linked Immunosorbent Assay; Rg: Radius of Gyration; SAXS: Small Angle X-ray Scattering; SE-HPLC: Size Exclusion High Performance Liquid Chromatography; SV-AUC: Sedimentation Velocity-Analytical Ultracentrifugation

The higher-order structure (HOS) of protein therapeutics has been confirmed as a critical quality parameter. In this study, we compared 2D 1H-13C ALSOFAST-HMQC NMR spectra with immunochemical ELISA-based analysis to evaluate their sensitivity in assessing the HOS of a potent human monoclonal antibody (mAb) for the treatment of coronavirus disease 2019 (COVID-19). The study confirmed that the methyl region of the 2D 1H-13C NMR spectrum is sensitive to changes in the secondary and tertiary structure of the mAb, more than ELISA immunoassay. Because of its highly detailed level of characterization (i.e., many 1H-13C cross-peaks are used for statistical comparability), the NMR technique also provided a more informative outcome for the product characterization of biopharmaceuticals. This NMR approach represents a powerful tool in assessing the overall higher-order structural integrity of mAb as an alternative to conventional immunoassays. [ABSTRACT FROM AUTHOR]

• An AEX method was developed for sgRNA as an alternative to IP-RPLC and HILIC. • Temperature-dependent AEX reveals the complex higher-order structure (HOS) of sgRNAs. • The HOS of sgRNA differs from other nucleic acids, providing additional chemical stability for sgRNA. Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs. [ABSTRACT FROM AUTHOR]

• Higher order structure characterization is critical to protein therapeutics development. • Recent advances in mass spectrometry have provided means to characterize HOS. • The utility of native MS, HDX-MS and FPOP-MS is presented in this review. • Future trend in MS applications to HOS characterization is described. Molecular characterization of higher order structure (HOS) in protein therapeutics is crucial to the selection of candidate molecules, understanding of structure–function relationships, formulation development, stability assessment, and comparability studies. Recent advances in mass spectrometry (MS), including native MS, hydrogen/deuterium exchange (HDX)-MS, and fast photochemical oxidation of proteins (FPOP) coupled with MS, have provided orthogonal ways to characterize HOS of protein therapeutics. In this review, we present the utility of native MS, HDX-MS and FPOP-MS in protein therapeutics discovery and development, with a focus on epitope mapping, aggregation assessment, and comparability studies. We also discuss future trends in the application of these MS methods to HOS characterization. [ABSTRACT FROM AUTHOR]

The higher-order structure (HOS) of protein therapeutics has been confirmed as a critical quality parameter. In this study, we compared 2D 1H-13C ALSOFAST-HMQC NMR spectra with immunochemical ELISA-based analysis to evaluate their sensitivity in assessing the HOS of a potent human monoclonal antibody (mAb) for the treatment of coronavirus disease 2019 (COVID-19). The study confirmed that the methyl region of the 2D 1H-13C NMR spectrum is sensitive to changes in the secondary and tertiary structure of the mAb, more than ELISA immunoassay. Because of its highly detailed level of characterization (i.e., many 1H-13C cross-peaks are used for statistical comparability), the NMR technique also provided a more informative outcome for the product characterization of biopharmaceuticals. This NMR approach represents a powerful tool in assessing the overall higher-order structural integrity of mAb as an alternative to conventional immunoassays.

Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprinting‐based approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein–protein interactions, and protein–ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev [ABSTRACT FROM AUTHOR]

Application of NMR spectroscopy to derive in-depth characterization of structure and dynamical properties of biomolecules is well established nowadays in many laboratories. Most of these methods rest on the availability of protein labeled with stable isotopes like 13C and 15N. In this report examples are presented on the application of NMR spectroscopic methods to characterize biopharmaceutical proteins in cases no isotope labeled material are available. This is typically found in protein samples used in the development of formulations and production processes. Another important focus of this report is the application of NMR methodology in the field of counterfeit drugs of biologicals and biosimilars. Especially here, NMR does offer relevant structural and quantitative data due to the high versatility of the NMR equipment. An excurse regarding the high medical relevance for a detailed spectroscopic analysis of counterfeits will be presented. [ABSTRACT FROM AUTHOR]

The present study examined the factor structure of the Differential Ability Scales--Second Edition (DAS-II) core subtests from the standardization sample via confirmatory factor analysis (CFA) using methods (bifactor modeling and variance partitioning) and procedures (robust model estimation due to nonnormal subtest score distributions) recommended but not included in the DAS-II Introductory and Technical Handbook. CFAs were conducted with the three DAS-II standardization sample age groups (lower early years [age = 2:6-3:5 years], upper early years [age = 3:6-6:11 years], school age [7:0-17:11 years]) using standardization sample raw data provided by NCS Pearson, Inc. Although most DAS-II core subtests were properly associated with the theoretically proposed group factors, both the higher order and bifactor models indicated that the g factor accounted for large portions of total and common variance, whereas the group factors (Verbal, Nonverbal, Spatial) accounted for small portions of total and common variance. The DAS-II core battery provides strong measurement of general intelligence, and clinical interpretation should be primarily, if not exclusively, at that level. [ABSTRACT FROM AUTHOR]

Community detection (or graph clustering) is crucial for unraveling the structural properties of complex networks. As an important technique in community detection, label propagation has shown the advantage of finding a good community structure with nearly linear time complexity. However, despite the progress that has been made, there are still several important issues that have not been properly addressed. First, the label propagation typically proceeds over the lower order structure of the network and only the direct one-hop connections between nodes are taken into consideration. Unfortunately, the higher order structure that may encode design principle of the network and be crucial for community detection is neglected under this regime. Second, the stability of the identified community structure may also be seriously affected by the inherent randomness in the label propagation process. To tackle the above issues, this article proposes a Motif-Aware Weighted Label Propagation method for community detection. We focus on triangles within the network, but our technique extends to other kinds of motifs as well. Specifically, the motif-based higher order structure mining is conducted to capture structural characteristics of the network. First, the motif of interest (locally meaningful pattern) is identified, and then, the motif-based hypergraph can be constructed to encode the higher order connections. To further utilize the structural information of the network, a re-weighted network is designed, which unifies both the higher order structure and the original lower order structure. Accordingly, a novel voting strategy termed NaS (considering both Number and Strength of connections) is proposed to update node labels during the label propagation process. In this way, the random label selection can be effectively eliminated, yielding more stable community structures. Experimental results on multiple real-world datasets have shown the superiority of the proposed method. [ABSTRACT FROM AUTHOR]

Poly(lactic acid) (PLA) has attracted much attention for use in bone fixation devices, which are used for fracture treatment, because it degrades to nontoxic lactic acid through nonenzymatic hydrolytic degradation. However, the application of such device is limited to relatively low-load regions, because mechanical properties of PLA is poor than that of metal materials. The process of drawing PLA fibers has attracted much attention as an approach for improving the mechanical properties of PLA bone fixation devices. The mechanical properties and molecular orientation of drawn PLA fibers and films have been investigated. However, the optimization of drawing conditions in bulk processes in relation to bone fixation devices is difficult because a large number of potential drawing conditions exist. The purpose of this study was to analytically clarify molecular orientation behavior in drawn PLA billets. The molecular orientation obtained by extrusion in PLA billets was investigated using a combination of the finite element method (FEM) and a chain network model. As a result of the FEM, first principal plastic strain was generated, mainly in the tapered components during extrusion; this strain was in the axial direction of the rod. Radius strain distributions showed that strain in the center region was almost uniform, whereas strain peaked near the surface. The orientation function increased with extrusion ratio (ER), and the peak orientation function in the surface region increased with taper angle. Thus, the magnitude and distribution of the orientation function in extruded PLA billets may be controllable by ER and taper angle, respectively.

The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out., (© 2023 The Authors. Proteomics published by Wiley-VCH GmbH.)

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior. [ABSTRACT FROM AUTHOR]

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. • The structural MS toolbox affords interesting methods for biosimilarity assessment. • MS approaches provides detection of small differences in biosimilar/originator mAbs. • Native MS coupled to size exclusion chromatography allows size variants evaluation. • Ion mobility spectrometry rapidly detects subtle changes in protein conformation. • HDX-MS is a key technique for higher order structure comparison of biosimilars. [ABSTRACT FROM AUTHOR]

The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15 N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.

Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics. [ABSTRACT FROM AUTHOR]

Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 Å in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 Å, although few contacts were observed for variable and CH3 regions.Heavy atom self contact maps of the NISTmAb indicate a highly-flexible structure. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy has a unique capability to probe the primary and higher order molecular structure and the structural dynamics of biomolecules at an atomic resolution, and this capability has been greatly fortified over the last five decades by an astonishing NMR instrumental and methodological development. Because of these factors, NMR has become a primary tool for the structure investigation of biomolecules, spawning a whole scientific subfield dedicated to the subject. This role of NMR is by now well established and broadly appreciated, especially in the context of academic research dealing with proteins that are purified and isotope-labeled in order to facilitate the necessary sophisticated multidimensional NMR measurements. However, the more recent industrial development, manufacturing, and quality control of biopharmaceuticals provide a different framework for NMR. For example, protein drug substances are not isotope-labeled and are present in a medium of excipients, which make structural NMR measurements much more difficult. On the other hand, biotechnology involves many other analytical requirements that can be efficiently addressed by NMR. In this respect the scope and limitations of NMR are less well understood. Having the non-expert reader in mind, herein we wish to highlight the ways in which modern NMR can effectively support biotechnological developments. Our focus will be on biosimilar proteins, pointing out certain cases where its use is probably essential. Based partly on literature data, and partly on our own hands-on experience, this paper is intended to be a guide for choosing the proper NMR approach for analytical questions concerning the structural comparability of therapeutic proteins, monitoring technology-related impurities, protein quantification, analysis of spent media, identification of extractable and leachable components, etc. Also, we focus on critical considerations, particularly those coming from drug authority guidelines, which limit the use of the well-established NMR tools in everyday practice. [ABSTRACT FROM AUTHOR]

Novel poly(thiophenyleneethynylenephenylene)s having optically inactive/active amide groups were synthesized by the Sonoghashira-Hagihara coupling polymerization of 3-substituted 2,5-dibromothiophene 1 with 1-substituted 3,5-diethynylbenzenes 2N , 2R , 2S derived from glycine and d -/ʟ-alanines. Poly( 1 – 2S ) exhibited an intense Cotton effect based on negative exciton chirality at 405 and 366 nm. On the other hand, poly( 1–2N ) exhibited a Cotton effect similar to that of poly( 1 – 2S ) but weaker, and poly( 1 – 2R ) exhibited a weak Cotton effect based on positive exciton chirality. The chiral secondary structures of the polymers were dependent on chirality cooperation and chirality competition of the side chains. The trend of the circular dichroism (CD) signs and intensities of the polymers can be explained by the concepts of “chiral cooperation” and “chiral competition” between the optically active thiophene unit and optically inactive/active phenylene unit. These polymers formed aggregate structures with increasing MeOH content of CHCl 3 /MeOH mixed solvents, with accompanying decrease of the UV–vis absorptions and photoluminescence (PL) intensities. [ABSTRACT FROM AUTHOR]

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1 H N - 15 N H heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR ‘cold probes.’ Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes. [ABSTRACT FROM AUTHOR]

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. [ABSTRACT FROM AUTHOR]

Characterization of the higher order structure (HOS) of protein-based biopharmaceutical products is an important aspect of their development. Opinions vary about how best to apply biophysical methods, in which contexts to use these methods, and how to use the resulting data to make technical decisions as drug candidates are commercialized [Gabrielson JP, Weiss WF IV. J Pharm Sci . 2015;104(4):1240-1245]. The aim of this commentary is to provide guidance for the development and implementation of a robust and comprehensive HOS characterization strategy. We first consider important concepts involved in developing a strategy that is appropriately suited to a particular biologic, and then discuss ways industry can partner with academia, technology companies, government laboratories, and regulatory agencies to improve the consistency with which HOS characterization is applied across the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

We have investigated the structure and activity of chicken egg-white lysozyme in aqueous solutions of two typical ionic liquids, 1-butyl-3-methylimidazolium chloride and ethylammonium nitrate, at cryogenic temperature. An increase in structural disorder due to the unfolding and a decrease in the α-helical structure of lysozyme were noticeable upon glass formation. However, a decrease in the structural stability after cooling was less than that before cooling. The secondary and tertiary structures showed good reversibility upon cooling to 77 K and then reverting back to ambient temperature. We discussed an influence of a cooling upon the structure in aqueous ionic liquid solutions. [ABSTRACT FROM AUTHOR]

Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development. [ABSTRACT FROM AUTHOR]

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope. [ABSTRACT FROM AUTHOR]

Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics. [ABSTRACT FROM AUTHOR]