Targeting product quality: Where systems biotechnology and process design meet

Product quality is a result of the entire production process including protein sequence, host cell, media and process parameters. Many of the desired product properties are defined by posttranslational modifications with impact on biological activity, immunogenicity, half-life or stability. In‑depth understanding of the host cells capabilities as well as of the process interactions enables the targeted modulation of product quality attributes by rational selection of host cells and design of bioprocesses. This is valuable for new biological molecules in order to improve efficacy, reduce side effects, access new patient populations. For biosimilars this allows developing into defined quality attribute profiles. The identification of suitable host cells, process parameters and media compositions to modulate quality attributes is challenging due to the complexity of the cell and the bio-processes. Here, we want to present two aspects of how we approach this challenge: First, by a global RNAseq-driven analysis that reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation (Könitzer & Müller et al., 2015) and second by comprehensive data analysis, in-depth characterization and high-throughput screening of process parameters and media compounds impacting glycosylation. To characterize different host cells a global analysis was performed on glyco-pattern and gene expression level. Six different monoclonal antibody projects with over 550 analyses were reviewed concerning their glyco-pattern distribution based on ESI-MS and HPLC data. Additionally, nearly 200 RNAseq gene expression data were used for a pathway-oriented analysis of the glycosylation-associated transcripts. Gene expression levels were compared between the three potential host cell lines as well as for host versus producing cell line. We identified with our new NGS pipeline 278 transcripts in our database. Expression patterns were host cell specific and depended on whether a mAb was expressed or not. For example, the expression of Sialyltransferase 10 (St3gal6) and B4galt6 (β 1,4-galactosyltransferase 6) could only be observed in the CHO-K1 host cell line while Cmah was only detectable in CHO-DG44 cells. Interestingly, St6gal1 was switched-on in mAb producing CHO-DG44 cells but at a very low level. this explains why normally only relatively low sialylation is observed with products produced in this cell line, and, since both the Sialyltransferase 10 and the CMP-Neu5Ac Hydroxylase activities are needed for constitution of with Neu5Gc sialic acid glycosylated antibodies, by lacking of the St3gal6 (CHO-DG44 cells) or the Cmah gene (CHO-K1 cells) mainly the non-immunogenic Neu5Ac sialic acids are predominant in CHO cells. Such data improve future production clone selection and process development strategies for better steering but may also support selection of critical quality attributes. The impact of cell culture conditions and media compounds on the glycosylation pattern was assessed by an integrated screening approach. Initially a database was created including process and analytical data from twelve projects. Data sets of more than 2500 fed-batch processes with 6300 analytical data sets enabled a cross-project analysis and correlation of process parameters with product quality attributes. Additionally, multi parallel small scale bioreactors, robotics based product capture and high throughput analytics were combined to minimize hands-on-time to gain data for correlation analysis. Said setups supported the identification of numerous media supplements and upstream process conditions that were applied for rational modulation of glycosylation patterns. Moreover, case studies focusing on the optimization of glycan patterns and antibody dependent cellular cytotoxicity by using metal ions as media supplements will be shown. Knowledge-driven selection of a host cell already gives direction to the product quality space to be expected with a certain molecule in clone selection. After gap analysis, process parameters can be chosen for application in process development to finally achieve the set quality target product profile

The Transient Accereting X-Ray Pulsar XTE J1946+274: Stability of the X-Ray Properties at Low Flux and Updated Orbital Solution

We present a timing and spectral analysis of the X-ray pulsar XTE J1946+274 observed with Suzaku during an outburst decline in 2010 October and compare with previous results. XTE J1946+274 is a transient X-ray binary consisting of a Be-type star and a neutron star with a 15.75 s pulse period in a 172 days orbit with 2–3 outbursts per orbit during phases of activity. We improve the orbital solution using data from multiple instruments. The X-ray spectrum can be described by an absorbed Fermi–Dirac cut-off power-law model along with a narrow Fe Kα line at 6.4 keV and a weak Cyclotron Resonance Scattering Feature (CRSF) at ~35 keV. The Suzaku data are consistent with the previously observed continuum flux versus iron line flux correlation expected from fluorescence emission along the line of sight. However, the observed iron line flux is slightly higher, indicating the possibility of a higher iron abundance or the presence of non-uniform material. We argue that the source most likely has only been observed in the subcritical (non-radiation dominated) state since its pulse profile is stable over all observed luminosities and the energy of the CRSF is approximately the same at the highest (~5 × 10^(37) erg s^(−1)) and lowest (~5 × 10^(36) erg s^(−1)) observed 3–60 keV luminosities

Tumour stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Tumour growth and metastatic infiltration are favoured by several components of the tumour microenvironment. Bone marrow-derived multipotent mesenchymal stromal cells (MSC) are known to contribute to the tumour stroma. When isolated from healthy bone marrow, MSC exert potent antiproliferative effects on immune effector cells. Due to phenotypic and morphological similarities of MSC and tumour stromal cells (TStrC), we speculated that immunotherapeutic approaches may be hampered if TStrC may still exhibit immunomodulatory properties of MSC.</p> <p>Methods</p> <p>In order to compare immunomodulatory properties of MSC and tumour stromal cells (TStrC), we established and analyzed TStrC cultures from eleven paediatric tumours and MSC preparations from bone marrow aspirates. Immunophenotyping, proliferation assays and NK cell cytotoxicity assays were employed to address the issue.</p> <p>Results</p> <p>While TStrC differed from MSC in terms of plasticity, they shared surface expression of CD105, CD73 and other markers used for MSC characterization. Furthermore, TStrC displayed a strong antiproliferative effect on peripheral blood mononuclear cells (PBMC) in coculture experiments similar to MSC. NK cell cytotoxicity was significantly impaired after co-culture with TStrC and expression of the activating NK cell receptors NKp44 and NKp46 was reduced.</p> <p>Conclusions</p> <p>Our data show that TStrC and MSC share important phenotypic and functional characteristics. The inhibitory effect of TStrC on PBMC and especially on NK cells may facilitate the immune evasion of paediatric tumours.</p

The ASSET Architecture - Integrating Media Applications and Products through a Unified API

International audienceApplications and products currently available for the broadcasting market are vertically integrated or proprietary. They are based on components requiring specific and costly development to interoperate and do rely typically on a single manufacturer or system integrator. Hence they are not fully compliant with broadcasters' requirements. ASSET is a European funded project whose main goal is to overcome the limitations of custom specific implementations of a digital system for TV content creation. These limitations are generally due to the misfit of interfaces between software layers, proprietary APIs of equipment from different vendors and the lack of a generalised middleware for multimedia content management with openly defined interfaces. Besides presenting the ASSET proposed architecture and concepts, this paper describes the prototype under development to test and demonstrate the project proposals

Tissue-specific regulatory network extractor (TS-REX): a database and software resource for the tissue and cell type-specific investigation of transcription factor-gene networks

The prediction of transcription factor binding sites in genomic sequences is in principle very useful to identify upstream regulatory factors. However, when applying this concept to genomes of multicellular organisms such as mammals, one has to deal with a large number of false positive predictions since many transcription factor genes are only expressed in specific tissues or cell types. We developed TS-REX, a database/software system that supports the analysis of tissue and cell type-specific transcription factor-gene networks based on expressed sequence tag abundance of transcription factor-encoding genes in UniGene EST libraries. The use of expression levels of transcription factor-encoding genes according to hierarchical anatomical classifications covering different tissues and cell types makes it possible to filter out irrelevant binding site predictions and to identify candidates of potential functional importance for further experimental testing. TS-REX covers ESTs from H. sapiens and M. musculus, and allows the characterization of both presence and specificity of transcription factors in user-specified tissues or cell types. The software allows users to interactively visualize transcription factor-gene networks, as well as to export data for further processing. TS-REX was applied to predict regulators of Polycomb group genes in six human tumor tissues and in human embryonic stem cells

Seismic structure of an oceanic core complex at the Mid-Atlantic Ridge, 22°19′N

We present results from a seismic refraction and wide-angle experiment surveying an oceanic core complex on the Mid-Atlantic Ridge at 22°19′N. Oceanic core complexes are settings where petrological sampling found exposed lower crustal and upper mantle rocks, exhumed by asymmetric crustal accretion involving detachment faulting at magmatically starved ridge sections. Tomographic inversion of our seismic data yielded lateral variations of P wave velocity within the upper 3 to 4 km of the lithosphere across the median valley. A joint modeling procedure of seismic P wave travel times and marine gravity field data was used to constrain crustal thickness variations and the structure of the uppermost mantle. A gradual increase of seismic velocities from the median valley to the east is connected to aging of the oceanic crust, while a rapid change of seismic velocities at the western ridge flank indicates profound differences in lithology between conjugated ridge flanks, caused by un-roofing lower crust rocks. Under the core complex crust is approximately 40% thinner than in the median valley and under the conjugated eastern flank. Clear PmP reflections turning under the western ridge flank suggest the creation of a Moho boundary and hence continuous magmatic accretion during core complex formation

Establishment of Fruit Bat Cells (Rousettus aegyptiacus) as a Model System for the Investigation of Filoviral Infection

Marburg virus and several species of Ebola virus are endemic in central Africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. So far, there is no vaccination or therapy available to protect people at risk in these regions. Recently, different fruit bats have been identified as potential reservoirs. One of them is Rousettus aegyptiacus. It seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral RNA, a phenomenon that is currently not understood. As a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. Here, we provide the first insights into this topic by monitoring filovirus infection of a Rousettus aegyptiacus derived cell line, R06E. We were able to show that filoviruses propagate well in R06E cells, which can, therefore, be used to investigate replication and transcription of filovirus RNA and to very efficiently perform rescue of recombinant Marburg virus using reverse genetics. These results emphasize the suitability of the newly established bat cell line for filovirus research

Hematopoietic Cell Transplantation Cures Adenosine Deaminase 2 Deficiency : Report on 30 Patients

Correction; Early Access: ' DOI: 10.1007/s10875-022-01280-y Early Access: APR 2022Purpose Deficiency of adenosine deaminase 2 (DADA2) is an inherited inborn error of immunity, characterized by autoinflammation (recurrent fever), vasculopathy (livedo racemosa, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency, lymphoproliferation, immune cytopenias, and bone marrow failure (BMF). Tumor necrosis factor (TNF-alpha) blockade is the treatment of choice for the vasculopathy, but often fails to reverse refractory cytopenia. We aimed to study the outcome of hematopoietic cell transplantation (HCT) in patients with DADA2. Methods We conducted a retrospective study on the outcome of HCT in patients with DADA2. The primary outcome was overall survival (OS). Results Thirty DADA2 patients from 12 countries received a total of 38 HCTs. The indications for HCT were BMF, immune cytopenia, malignancy, or immunodeficiency. Median age at HCT was 9 years (range: 2-28 years). The conditioning regimens for the final transplants were myeloablative (n = 20), reduced intensity (n = 8), or non-myeloablative (n = 2). Donors were HLA-matched related (n = 4), HLA-matched unrelated (n = 16), HLA-haploidentical (n = 2), or HLA-mismatched unrelated (n = 8). After a median follow-up of 2 years (range: 0.5-16 years), 2-year OS was 97%, and 2-year GvHD-free relapse-free survival was 73%. The hematological and immunological phenotypes resolved, and there were no new vascular events. Plasma ADA2 enzyme activity normalized in 16/17 patients tested. Six patients required more than one HCT. Conclusion HCT was an effective treatment for DADA2, successfully reversing the refractory cytopenia, as well as the vasculopathy and immunodeficiency. Clinical Implications HCT is a definitive cure for DADA2 with > 95% survival.Peer reviewe