Development and application of glycosyltransferases for in vitro glycoengineering

Glycosylation is an important posttranslational modification of proteins influencing protein folding, stability and regulation of the biological activity, e.g. IgG featuring terminal sialic acids were shown to suppress inflammatory response (ADCC) and increase serum half-life. Fully galactosylated structures seem to improve binding to the complement system (CDC). Despite all efforts in cell-line engineering and process optimization to increase glycosylation homogeneity, today therapeutic proteins still show a heterogeneous glycosylation pattern, with large variations between bioprocesses and even from batch to batch. The use of glycosyltransferases for enzymatic synthesis of well-defined glycan structures will become an essential tool – at least in order to provide clear conclusions on the structure-function relationship of different glycan variants. Therefore, highly active derivatives of human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-1), beta-1,4 galactosyltransferase 2 (B4Gal-T2), and beta-galactoside alpha-2,3 sialyltransferase 6 (ST3Gal-6) are currently developed for secreted expression by transient gene expression (TGE) as well as CHO-K1 for future GMP production. To our surprise, N-terminally truncated variants of human ST6Gal-1 allow directed G2+1SA and G2+2SA mab glycoengineering in sialylation experiments using bi-antennary glycans (mabs) as well as tetra-antennary glycans (EPO) as substrate: The Δ108 variant produces mainly mono-sialylated N-glycans. In contrast, Δ89 – which is commercially available – produces a high degree of bi-sialylated glycans during the first 8 hours followed by sialydase activity leading to a continuously decreasing overall sialylation level. Consequently, dependent on the incubation time different sialylation patterns can be achieved. The x-ray structure of the Δ89 variant is described in Kuhn et al. (2013) Acta Crystallography 69:1826-38. We used in vitro glycoengineering (IVGE) to investigate the impact of IgG1 Fc glycans on effector functionality: After creating a unique set of Fc glycan variants of an IgG1 with different levels of galactose and sialic acid, respectively, we analyzed their impact on FcγRI, IIa and IIIa binding by Surface Plasmon Resonance (SPR), and on ADCC activity. Furthermore, we supported an early stage project by IVGE to prepare material for mouse PK studies

Proteolysis of non-IgG molecules in transient HEK293 and stable CHO-K1 bioprocesses

Since 2006, we use the transient FreeStyle™ 293 System (Thermo Fisher Scientific) as well as an in-house developed stable CHO-K1 platform in order to express recombinant molecules for antibody generation and prototype assay development. Successively, more than 200 different human antigens – secreted and cytosolic – were cloned, expressed, purified via Ni-chelate chromatography and analyzed by SDS-PAGE, UV scan, analytical size exclusion (HPLC/RALS) and dynamic light scattering (DLS). A major bioprocess issue can be proteolytic degradation, also referred as ‘clipping’, by proteases originating from the host cells, thereby leaving an altered, non- or less functional protein of interest. We experience that approx. 5% of our human non-IgG target molecules are prone to clipping. The use of protein/serum-free chemically defined media in both platforms might also trigger the proteolysis of the protein of interest. So far, this issue was poorly addressed by the scientific community, in part since IgG antibodies – which receive the most attention – tend to be well expressed, robust, and stable molecules. Little is known about the proteases expressed by the host, and about the proteases responsible for clipping. Another challenge is the sheer number of proteases since 100s proteases are known to be present in cell genomes and may be involved in the clipping process. Outline of the presentation: 1. Examples of HEK- and CHO-derived molecules are presented with their cleavage sites identified by Edman degradation following SDS-PAGE separation and blotting. In general, the proteolysis is not quantitative – rather, a distinct fragmentation ladder is observed by SDS-PAGE analysis. Unfortunately, the cleavage sites do not follow a strict pattern indicating that several different proteases might be in charge, depending on the host cell line as well. 2. Analysis of HEK- and CHO-supernatants by gelatin and caseine zymography reveal the activity of several distinct proteases for each host cell line. 3. The Human Protease Array Kit (R&D Systems) reveils a broad panel of highly expressed cathepsins and metalloproteinases in HEK293-supernatants. 4. Experiments utilizing siRNA as well as co-expression of inhibitor molecules are currently performed in order to track down (and hopefully inhibit) the responsible proteases for each bioproces

Motif Discovery in Tissue-Specific Regulatory Sequences Using Directed Information

Motif discovery for the identification of functional regulatory elements underlying gene expression is a challenging problem. Sequence inspection often leads to discovery of novel motifs (including transcription factor sites) with previously uncharacterized function in gene expression. Coupled with the complexity underlying tissue-specific gene expression, there are several motifs that are putatively responsible for expression in a certain cell type. This has important implications in understanding fundamental biological processes such as development and disease progression. In this work, we present an approach to the identification of motifs (not necessarily transcription factor sites) and examine its application to some questions in current bioinformatics research. These motifs are seen to discriminate tissue-specific gene promoter or regulatory regions from those that are not tissue-specific. There are two main contributions of this work. Firstly, we propose the use of directed information for such classification constrained motif discovery, and then use the selected features with a support vector machine (SVM) classifier to find the tissue specificity of any sequence of interest. Such analysis yields several novel interesting motifs that merit further experimental characterization. Furthermore, this approach leads to a principled framework for the prospective examination of any chosen motif to be discriminatory motif for a group of coexpressed/coregulated genes, thereby integrating sequence and expression perspectives. We hypothesize that the discovery of these motifs would enable the large-scale investigation for the tissue-specific regulatory role of any conserved sequence element identified from genome-wide studies

High-speed CH planar laser-induced fluorescence imaging using a multimode-pumped optical parametric oscillator

We report on high-speed CH planar laser-induced fluorescence (PLIF) imaging in turbulent diffusion flames using a multimode-pumped optical parametric oscillator (OPO). The OPO is pumped by the third-harmonic output of a multimode Nd:YAG cluster for direct signal excitation in the A-X (0,0) band of the CH radical. The lasing threshold, conversion efficiency, and linewidth are shown to depend on the number of pump passes in the ring cavity of the OPO. Single-shot CH PLIF images are acquired at 10 kHz with excitation energy up to 6 mJ=pulse at 431:1nm. Signalto- noise ratios of ∼25–35 are the highest yet reported for high-speed CH PLIF

An Empirical Survey on Co-simulation: Promising Standards, Challenges and Research Needs

Co-simulation is a promising approach for the modelling and simulation of complex systems, that makes use of mature simulation tools in the respective domains. It has been applied in wildly different domains, oftentimes without a comprehensive study of the impact to the simulation results. As a consequence, over the recent years, researchers have set out to understand the essential challenges arising from the application of this technique. This paper complements the existing surveys in that the social and empirical aspects were addressed. More than 50 experts participated in a two-stage Delphi study to determine current challenges, research needs and promising standards and tools. Furthermore, an analysis of the strengths, weakness, opportunities and threats of co-simulation utilizing the analytic hierarchy process resulting in a SWOT-AHP analysis is presented. The empirical results of this study show that experts consider the FMI standard to be the most promising standard for continuous time, discrete event and hybrid co-simulation. The results of the SWOT-AHP analysis indicate that factors related to strengths and opportunities predominate