Equal mixing time enables scale-down and optimization of a CHO cell culture process using a shaken microbioreactor system

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. The monitoring and control capabilities of microbioreactors not only promote the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool. Using the Dual Indicator System for Mixing Time, the system was initially characterized for mixing properties at varying operating conditions, which was found to yield mixing times between 0.9 and 41.8 s. A matched mixing time was proposed as scale-down criterion for an IgG4 producing GS-CHO fed-batch process between a 5 L stirred tank reactor (STR) and the micro-Matrix microbioreactor. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale-down model was then employed to optimize a bolus feeding regime using response surface methodology, which led to a 25.4% increase of the space-time yield and a 25% increase of the final titre. The optimized feeding strategy was validated at the small-scale and successfully scaled up to the 5 L STR. This work for the first time provides a framework of how the micro-Matrix microbioreactor can be implemented in a bioprocess development workflow and demonstrates scalability of growth and production kinetics as well as IgG4 glycosylation between the micro-Matrix and a benchtop-scale STR system. Graphical Abstract and Lay Summary: (Figure presented.) Microbioreactor technology has become an essential part of early- and mid-stage bioprocess development. This study provides a framework of how the shaken micro-Matrix system can be used for cell culture process development of a mammalian CHO cell line producing a monoclonal antibody. Following scale-down from a 5 L stirred tank bioreactor, the micro-Matrix was employed for the optimisation of a feeding strategy and the optimised protocol was successfully scaled up

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease

Glycan microheterogeneity at the PGT135 antibody recognition site on HIV-1 gp120 reveals a molecular mechanism for neutralization resistance.

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design

Common security and defense policy – the way ahead

info:eu-repo/semantics/publishedVersio

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown

\ua9 2020, The Author(s).The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states

Composition and antigenic effects of individual glycan sites of a trimeric HIV-1 envelope glycoprotein

The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens