Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

Leadership position and physician visits – results of a nationally representative longitudinal study in Germany

Background

So far, studies within the occupational field have largely concentrated on working conditions and job stressors and staff members’ or subordinate health. Only a few have focused on managers in this context, but studies are missing that explicitly look at the relation between leadership position and health care use (HCU). Thus, the purpose of this study was to examine the potential effects of a change in leadership position on HCU in women and men longitudinally.

Methods

Data were drawn from a nationally representative longitudinal study in Germany (German Socio-Economic Panel, GSOEP). Data from 2009 and 2013 were used. Leadership position was divided into (i) top management, (ii) middle management, (iii) lower management, and (iv) a highly qualified specialist position. The number of physician visits in the preceding 3 months were used to quantify HCU (n?=?2140 observations in regression analysis; 69% male).

Results

Adjusting for various potential confounders (e.g., age, self-rated health, chronic conditions, and personality factors), Poisson FE regression analysis revealed that changes from a highly qualified specialist position to the top management were associated with a decrease in the number of physician visits in men (β?=?.47, p?<?.05), but not in women. Gender differences (gender x leadership position) were significant.

Conclusions

Findings of this study emphasize the impact of leadership positions on the number of physician visits in men. Further study is required to elucidate the underlying mechanisms.

     

Mapping air pollutants at municipality level in Italy and Spain in support to health impact evaluations

A growing health concern, due to poor air quality, recently led to an increased number of studies regarding air pollution effects on public health. Consequently, close attention is paid to estimation methods of exposure to atmospheric pollutants. This paper aims to meet a specific requirement of epidemiological researchers, that is providing annual air pollution maps at municipality scale for health impact assessment purposes on national basis. Firstly, data fusion through kriging with external drift is implemented, combining pollution data from two different sources, models and measurements, in order to improve the spatial distribution of surface concentrations at grid level. Then, the assimilated data of air pollution are upscaled, so as to obtain concentrations at municipality level. This methodology was applied to Italy and Spain (in Spain, only the second step was carried out since the modeled concentration already included an assimilation procedure). In both countries, for each municipality, an estimate of the concentration value for atmospheric pollutants of major concern for human health (PM₁₀ and NO₂) was provided, offering more relevant information from a surveillance point of view.     

Toluene and methylethylketone: effect of combined exposure on their metabolism in rat

1.?Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK).

2.?Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated.

3.?Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations.

4.?In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.     

Delayed Urinary Symptoms Induced by Ketamine

One of the side-effects of ketamine abuse is genito-urinary damage. This report describes a case of a former ketamine user who presented with urinary symptoms associated with ketamine years after stopping consumption. This was a 26-year-old male with a history of ketamine abuse. He started treatment for alcohol dependence at age 19. He smoked marijuana daily and denied any other drug use. During the follow-up, urinary symptoms were evidenced (dysuria, frequency, urgency, incontinence, nocturia, hematuria, and suprapubic pain). Urinary symptoms started two years ago and worsened over time. The patient was referred to a urologist. A cystoscopy revealed lesions compatible with interstitial cystitis like the ones that appear in some ketamine abusers. Given the medical history, the urologist asked him about ketamine consumption and the patient declared a daily use of 50 milligrams intranasally from age 15 to age 17. Given these findings, not reported previously in the medical literature, future research should follow up patients who at some point in their life made an abusive consumption of ketamine in order to understand the pathogenesis and to be able to intervene before clinical disease manifests itself.     

Mitigation of Oxidation in Therapeutic Antibody Formulations: a Biochemical Efficacy and Safety Evaluation of <Emphasis Type="Italic">N</Emphasis>-Acetyl-Tryptophan and L-Methionine

Purpose

Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs.

Methods

Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine.

Results

Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients.

Conclusions

N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.