Objective
Glycation is a non-enzymatic and spontaneous post-translational modification (PTM) generated by the reaction between reducing sugars and primary amine groups within proteins. Because ...glycation can alter the properties of proteins, it is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) and should therefore be carefully monitored. The most abundant product of glycation is formed by glucose and lysine side chains resulting in fructoselysine after Amadori rearrangement. In proteomics, which routinely uses a combination of chromatography and mass spectrometry to analyze PTMs, there is no straight-forward way to distinguish between glycation products of a reducing monosaccharide and an additional hexose within a glycan, since both lead to a mass difference of 162 Da.
Methods
To verify that the observed mass change is indeed a glycation product, we developed an approach based on 2D NMR spectroscopy spectroscopy and full-length protein samples denatured using high concentrations of deuterated urea.
Results
The dominating β-pyranose form of the Amadori product shows a characteristic chemical shift correlation pattern in 1H-13C HSQC spectra suited to identify glucose-induced glycation. The same pattern was observed in spectra of a variety of artificially glycated proteins, including two mAbs, as well as natural proteins.
Conclusion
Based on this unique correlation pattern, 2D NMR spectroscopy can be used to unambiguously identify glucose-induced glycation in any protein of interest. We provide a robust method that is orthogonal to MS-based methods and can also be used for cross-validation.
The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy ...heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8-86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.
Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional ...bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.
Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. ...These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.
Court-mandated treatments imply a dual role for therapy providers not only of caring for, but also of having control over, involuntary clients. The impact of legal coercion on the therapeutic ...relationship and feelings of stigma is widely regarded as negative and detrimental for treatment outcomes. This point of view stands in contrast to advocates of the perspective that involuntary treatment can ameliorate social functioning and thus promote a better quality of life. Regarding other outcome measures, there is evidence that offender treatment is effective and leads to reduced recidivism in criminal behavior. This narrative review provides an overview of research assessing the effects of mandatory treatment on therapeutic process and outcome factors. We conclude that legal mandatory treatment does not have to necessarily result in perceived coercion and reduced satisfaction with treatment and that a caring and authoritative treatment style aids a favorable therapeutic alliance, motivation, and therapy outcomes.
Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and ...efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC-MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone,
-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.
In acute psychiatry, where people with severe mental disorders are frequently treated, there can be contradictions between concepts of illness among, e.g., patients and healthcare professionals, and ...also between medical and legal aspects. These contradictions do not manifest themselves openly but are immanent in the social practices of the treatment teams as contradictions between the social level and the individual level. They can lead to alienation, which may be reflected in poorer quality of treatment, such as the more frequent use of coercive measures or poorer adherence to therapy in patients. In the normal daily routine of a clinic, these contradictions are mostly hidden by hierarchical structures or by unbalanced concepts of psychiatric illness, or external critique is used to try to solve these contradictions. However, another way of dealing with these contradictions could be to analyze the potential and causes for alienation through systematic analysis and transformation of the whole system of a psychiatric ward to reduce the level of contradiction within it. The aim of this work is to use the concept of meaning elaborated by Luhmann to identify and recognize alienation potentials as concretely as possible and thus make them accessible to immanent critique. Meaning in Luhmann's use of the term serves to reduce complexity in a social context and always opens up consequential possibilities for action. Consequential limited possibilities at the level of action in a rigid social system (which psychiatric wards can be) can-at an individual level-lead to people subordinating themselves to the rigid system to an excessive degree and thus alienating themselves from the system. Thus, a rigid system with a narrowing of consequential possibilities excludes meaningful consequential possibilities. This leads to alienating contradictions and to possibilities of world appropriation being missed. The aim of the current analysis is not to make a general critique of psychiatry but to improve the theoretical basis to better understand the problem of alienation in acute psychiatry as a symptom of system-immanent contradictions and thus open up the possibility of transforming systems, e.g., psychiatric acute care units, by means of immanent critique.
High utilizers (HU) are patients with an above-average use of psychiatric inpatient treatment. A precise characterization of this patient group is important when tailoring specific treatment ...approaches for them. While the current literature reports evidence of sociodemographic, and socio-clinical characteristics of HU, knowledge regarding their psychological characteristics is sparse. This study aimed to investigate the association between patients' psychological characteristics and their utilization of psychiatric inpatient treatment. Patients from the University Psychiatric Clinics (UPK) Basel diagnosed with schizophrenia spectrum or bipolar affective disorders participated in a survey at the end of their inpatient treatment stay. The survey included assessments of psychological characteristics such as quality of life, self-esteem, self-stigma, subjective experience and meaning of psychoses, insight into the disease, and patients' utilization of psychiatric inpatient treatment in the last 30 months. The outcome variables were two indicators of utilization of psychiatric inpatient treatment, viz. "utilization pattern" (defined as HU vs. Non-HU NHU) and "length of stay" (number of inpatient treatment days in the last 30 months). Statistical analyses included multiple regression models, the least absolute shrinkage and selection operator (lasso) method, and the random forest model. We included 112 inpatients, of which 50 were classified as HU and 62 as NHU. The low performance of all statistical models used after cross-validation suggests that none of the estimated psychological variables showed predictive accuracy and hence clinical relevance regarding these two outcomes. Results indicate no link between psychological characteristics and inpatient treatment utilization in patients diagnosed with schizophrenia spectrum or bipolar affective disorders. Thus, in this study, the examined psychological variables do not seem to play an important role in patients' use of psychiatric inpatient treatment; this highlights the need for additional research to further examine underlying mechanisms of high utilization of psychiatric inpatient treatment.
Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a ...non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d
4
in D
2
O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.
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