Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - mass spectrometry

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V.Proteomic

Death anxiety resilience; a mixed methods investigation

Research was conducted examining how death anxiety influenced PTSD and mental health among people who have experienced a life-threatening event. This study was conducted using undergraduate university students in Lithuania. The study used a mixed-method design and in phase 1, participants (N = 97) completed self-report questionnaires that gathered information on demographics, death anxiety, trauma and well-being. Data indicated a significant correlation between death anxiety and PTSD, but not psychiatric co-morbidity. Phase 2 attempted to further explore the phenomenological experience of participants with full PTSD, and 6 semi-structured interviews were conducted. IPA analysis found three major themes in response to the life-threatening event; self-efficacy, religious coping and existential attitude. Overall these coping mechanisms allowed participants to develop resilience against the effects of death anxiety and minimize its negative impact on mental health

Estimation of stops' spectral place cues using multitaper techniques Estimação das características espectrais relacionadas com o ponto de articulação de oclusivas utilizando a técnica multitaper

This study focuses on the spectral characteristics of the European Portuguese stops /p, b, t, d, k, g/ produced by six native speakers. We analysed the spectral peaks and troughs by means of multitaper spectra and performed a parameterisation of the stop spectra using slope and moment measures. In comparison to traditional spectral estimations, multitaper is more exact and, more importantly, not limited to a stationary signal length necessary for the analysis window. Therefore, it is well-suited for the rather short duration of the burst of a stop. Results show that the burst characteristics vary with place of articulation. While the global spectral frequencies match the data in classical literature, it is shown that other spectral measures in our data do not follow the typical classical spectral patterns. It is discussed whether these differences are due to the use of different methodology, or substantial cross-linguistic differences in the spectral characteristics.<br>Este estudo analisa as caraterísticas espetrais das oclusivas /p, b, t, d, k, g/ do Português Europeu produzidas por seis informantes nativos. Procedeu-se à análise dos picos e dos vales espetrais (espetros multitaper) e à parametrização das caraterísticas espetrais através da análise dos declives dos espetros e do cálculo dos momentos de distribuição. Comparativamente às análises espetrais tradicionais, a análise multitaper permite um maior controlo da variância associada a este tipo de sinais fazendo uso de apenas uma curta janela, o que se adequa às caraterísticas do burst das oclusivas. Os resultados mostram que as caraterísticas do burst variam de acordo com o ponto de articulação. As frequências espetrais obtidas correspondem aos resultados publicados na literatura clássica. No entanto, verifica-se que outras medidas espetrais, no presente estudo, não estão de acordo com os padrões espetrais clássicos. É discutido se estas diferenças se devem ao uso de diferentes metodologias, ou a diferenças substanciais nas caraterísticas espetrais das diferentes línguas