Traces matter: Targeted optimization of monoclonal antibody N-glycosylation based on/by implementing automated high-throughput trace element screening

The use of high-throughput systems in cell culture process optimization offers various opportunities in biopharmaceutical process development. Here we describe the potential for acceleration and enhancement of product quality optimization and de novo bioprocess design regarding monoclonal antibody N-glycosylation by using an iterative statistical Design of Experiments (DoE) strategy based on our automated microtiter plate-based system for suspension cell culture. In our example, the combination of an initial screening of trace metal building blocks with a comprehensive DoE-based screening of 13 different trace elemental ions at three concentration levels in one run revealed most effective levers for N-glycan processing and biomass formation. Obtained results served to evaluate optimal concentration ranges and the right supplementation timing of relevant trace elements at shake flask and 2 L bioreactor scale. This setup identified manganese, copper, zinc, and iron as major factors. Manganese and copper acted as inverse key players in N-glycosylation, showing a positive effect of manganese and a negative effect of copper on glycan maturation in a zinc-dependent manner. Zinc and iron similarly improved cell growth and biomass formation. These findings allowed determining optimal concentration ranges for all four trace elements to establish control on desired product quality attributes regarding premature afucosylated and mature galactosylated glycan species. Our results demonstrates the power of combining robotics with DoE screening to enhance product quality optimization and to improve process understanding, thus, enabling targeted product quality control.     

Fine grinding or expanding of feed as pre-treatment for pelleting in dependence on dietary rapeseed expeller proportion: Nutritional consequences for broilers

Although commercial broiler feed is usually differently conditioned before pelleting, the nutritional consequences of fine grinding or expanding as pre-pelleting treatments are poorly defined so far. Therefore, the effects of these two pre-treatments on nutrient digestibility, broiler performance and digestive tract of broilers were tested. In order to investigate possible interactions between pre-treatments and diet composition two diets differing in rapeseed expeller proportion were tested in a two by two factorial design. Thus, four diets were designed including two diets containing 6% rapeseed expeller (RSE) which were pre-treated by fine grinding (6%FgP) or expanding (6%ExP), and two corresponding diets containing 12% RSE (12%FgP and 12%ExP). For the experiments, 864 male broilers were used. There was a significant diet-by-technical feed treatment (TFT) interaction in case of the digestibility of all considered crude nutrients (p < 0.05). Diet 6%ExP showed higher crude protein digestibility compared to other feeds (p < 0.001). The highest digestibility of organic matter, ether extract, crude fibre and N-free extractives achieved diet 12%FgP. Diets 6%ExP and 12%FgP showed higher N-corrected metabolisable energy content (p < 0.001). TFT affected daily feed intake (DFI) and body weight (BW) gain in a diet-dependent manner (p < 0.001). Feeding of 6%FgP enhanced DFI and BW gain compared to other feeds but 6%ExP reduced both parameters (p < 0.001). Weights of proventriculi and gizzards of animals fed 6%ExP were increased compared with 6%FgP (p < 0.01). In contrast, proventricular length in animals fed 6%FgP was increased compared with diet 6%ExP (p = 0.042). Moreover, animals fed 6%FgP had wider proventriculi than animals fed 12%FgP (p = 0.023). Feed 6%ExP increased proventricular weight compared to 12%ExP (p = 0.001). With regard to the strong relationships between diet and TFT no specific processing method can be recommended according to considered nutritional aspects. A marked prevention of proventricular dilatation due to pellet feeding could not be realised by various used TFT or feed formulations. Used amounts of RSE had no obvious adverse effects on considered nutritional aspects.     

Proteomic responses to ocean acidification of the marine diazotroph Trichodesmium under iron-replete and iron-limited conditions

Photosynthesis Research - Growth and dinitrogen (N2) fixation of the globally important diazotrophic cyanobacteria Trichodesmium are often limited by iron (Fe) availability in surface seawaters. To...     

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.     

Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination?

We applied real-time broad-range polymerase chain reaction (PCR) to detect bacteraemia in blood from febrile patients. Interpretation of amplification results in relation to clinical data and blood culture outcome was complex, although the reproducibility of the PCR results was good. Sequencing analysis of the PCR products revealed the presence of Burkholderia species DNA while no Burkholderia species grew in culture. The source of this contamination was shown to be the commercial DNA isolation kit used in the automated MagNA Pure Isolation Robot. A high degree of suspicion is required when uncommon or unexpected pathogens are diagnosed by molecular methods as clinical consequences can be serious.     

Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.     

Trimethyllysine,vascular risk factors and outcome in acute ischemic stroke (MARK–STROKE)

Trimethyllysine (TML) is involved in the generation of the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO) by gut microbiota. In clinical studies, elevated TML levels predicted major adverse cardiovascular events (MACE) in patients with acute or stable coronary artery disease (CAD). In contrast to cardiovascular patients, the role of TML in patients with acute cerebral ischemia is unknown. Here, we evaluated circulating TML levels in 374 stroke patients from the prospective biomarkers in stroke (MARK–STROKE) study. Compared with 167 matched healthy controls, acute ischemic stroke patients had lower median TML plasma concentrations, i.e. 0.71 vs. 0.47 µmol/L (p < 0.001) and this difference persisted after adjusting for age and sex. TML plasma concentrations were associated with age, serum creatinine, glucose, cholesterol and lysine. Patients with prevalent arterial hypertension, atrial fibrillation or a history of myocardial infarction had increased TML levels, but this observation was not independent of age, sex and GFR. In 274 patients, follow-up data were available. During a median follow-up of 284 [25th–75th percentile: 198, 431] days, TML was not associated with incident MACE (stroke, myocardial infarction, death). In summary, our data suggests a different role of TML in acute ischemic stroke compared with CAD patients.

     

Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.     

ARIADNE’S THREAD: GUIDING A PROTEIN ACROSS FIVE MEMBRANES IN CRYPTOPHYTES1

Cryptophytes are the most archetypal chromalveolates, with their complex plastid having retained many features of the red algal secondary endosymbiont. Most important of these is the remnant nucleus, the nucleomorph, that is kept between the inner and outer membrane pair of the endosymbiont in the highly reduced cytosol, the periplastidial compartment (PPC). Because the nucleomorph’s coding capacity is very limited, proteins need to be imported from the host cytosol across the outer two membranes into the PPC and across all four membranes into the stroma. How this is accomplished has puzzled researchers for >20 years. Recent findings show that in both cases, a bipartite topogenic signal, a signal and subsequent transit peptide (TP), is responsible for targeting proteins correctly into these two compartments. An aromatic amino acid–based motif at the +1 position of the TP holds the information determining into which compartment the precursor protein is finally transported. Together with the identification of a novel endoplasmic reticulum associated degradation (ERAD)–derived translocon in the second‐outermost membrane, these findings help us to understand the sophisticated targeting mechanisms across four membranes and clarify a key innovation during chromalveolate evolution.