Pseudo-affinity purification and formulation of a cell-culture derived whole influenza virus vaccine using magnetic sulfated cellulose particles

The production of viral vaccines usually employs different unit operations where formulation and filling are the final steps of downstream processing (DSP). However, complex DSP is often hard to realize in research laboratories focusing on novel vaccine candidates. Moreover, there are no real ready-to-use tools for high-throughput DSP of whole virus particles that can speed up development. Because of these needs we developed a new platform for easy and straightforward whole virus particle purification and formulation based on magnetic sulfated cellulose particles (MSCP)1,2. Proof of concept was carried out with an influenza A/Puerto Rico/8/34 (H1N1) whole virus vaccine for the immunization of mice. The virus particles were produced in suspension MDCK cells, clarified, inactivated, and concentrated using a standard protocol. After diafiltration to low salt buffer, the virus particles were bound to the MSCP and the virus loaded MSCP were washed and resuspended in formulation buffer. Please click Additional Files below to see the full abstract

Intensification of MVA and influenza virus production through high-cell-density cultivation approaches

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is often constrained by a decrease in cell-specific virus yields, early host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve high-yield virus production processes. In this study, the development of a semi-continuous process for the production of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza virus A/PR/8/34 (H1N1) in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented. Methods. Depending on the required scale, high cell concentrations (~ 50×106 cells/mL) were achieved either through medium renewal by periodic centrifugation (semi-perfusion) in 50 mL cultivations or using an alternating tangential flow (ATF) perfusion system for 1 L bioreactors. Process development and optimization comprised three phases: 1) assessment of different fed-batch and medium exchange strategies for the propagation of MVA-CR19 or influenza A/PR/8/34 viruses in 50 mL cultivations; 2) scale-up and process optimization of the selected high-yield process strategy to a 1 L bioreactor with the ATF system, and 3) integration of a one-step purification process using magnetic sulfated cellulose particles (MSCP). For both viruses, conventional batch cultivation (no addition/medium exchange after infection) was compared with processes applying fed-batch, periodic medium exchange and the combination of both during virus propagation. Results. Perfusion and semi-perfusion at a feeding rate of 0.05 nL/cell×d was suitable to propagate AGE1.CR.pIX cells above 60×106 cells/mL with neither limitation nor overload of nutrients. For infections at 50 mL scale, the application of a combined strategy comprising an initial fed-batch phase followed by a periodic virus harvest phase resulted in the highest product yield with a more than 10-fold increase in virus particles concentration compared to the conventional batch processes operated at 4 to 8×106 cells/mL [2]. Additionally, a 3-fold increase in both cell-specific yield (virus particles/cell) and volumetric productivity (virus particles/L×d) could be obtained. Comparable yields were observed when up-scaling to a 1 L bioreactor using an ATF-system, even when virus particles were retained within the bioreactor. Further selection of the optimal pore size of the ATF membrane allowed semi-continuous harvesting of the produced viruses and its purification with MSCPs with a recovery from 30 to 50%. In all cases, cell-specific yields and volumetric productivities reached their maxima at 72 h post-infection, indicating that the process should be stopped at that time point. Conclusion. Compared to conventional batch processes, the developed HCD process offers significantly higher productivities including the option to integrate a one-step purification process in a semi-continuous mode. Overall, the results show that there is a great potential for semi-continuous HCD processes for the production of viral vaccines in larger scales, which could support efforts towards the establishment of continuous vaccine manufacturing. References. 1. Pohlscheidt, M., et al., Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium. Vaccine, 2008. 26(12): p. 1552-65. 2. Lohr, V., et al., New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: studies on growth, metabolism and virus propagation. Vaccine, 2009. 27(36): p. 4975-82

14 Examples of How LLMs Can Transform Materials Science and Chemistry: A Reflection on a Large Language Model Hackathon

Chemistry and materials science are complex. Recently, there have been great successes in addressing this complexity using data-driven or computational techniques. Yet, the necessity of input structured in very specific forms and the fact that there is an ever-growing number of tools creates usability and accessibility challenges. Coupled with the reality that much data in these disciplines is unstructured, the effectiveness of these tools is limited. Motivated by recent works that indicated that large language models (LLMs) might help address some of these issues, we organized a hackathon event on the applications of LLMs in chemistry, materials science, and beyond. This article chronicles the projects built as part of this hackathon. Participants employed LLMs for various applications, including predicting properties of molecules and materials, designing novel interfaces for tools, extracting knowledge from unstructured data, and developing new educational applications. The diverse topics and the fact that working prototypes could be generated in less than two days highlight that LLMs will profoundly impact the future of our fields. The rich collection of ideas and projects also indicates that the applications of LLMs are not limited to materials science and chemistry but offer potential benefits to a wide range of scientific disciplines

Development of an SNP application based on real-time high resolution melting analysis of surface immobilized nucleic acids

Das Ziel dieser Masterarbeit war es, eine Real-Time Microarray-basierte SNP-Anwendung zu entwickeln. Die SNP-Diskriminierung wird durch Real-Time-Analyse, Hybridisierungskinetik und Schmelzkurven-Analyse, mittels immobilisierter DNA auf dem Microarray und DNA in Lösung erreicht. Eine der großen Herausforderungen bei der Real-Time Messung ist die Aufbereitung der umfangreichen Daten, die aus jeder Messung generiert werden: Die Messdaten von ca. 1100 Spots werden dabei in ein selbst entwickeltes MATLAB®-Programm geladen, wo diese dann sortiert, normalisiert und gefittet werden. Anschließend werden die Kinetiken und Schmelzvorgänge graphisch als Diagramm und die Fitting-Parameter in zusammenfassender Form ausgegeben. Für das Probe-Design wurde ein MATLAB®-Programm entwickelt, welches die SNP-Microarray-Probes basierend auf den sequenzierten Gen-Abschnitten des biologischen Materials automatisch ermittelt. Um dies zu bewerkstelligen, werden zuerst die SNP-Positionen in den zwei Spezien identifiziert. Dann werden die potentiellen Probe-Sequenz-Varianten, die durch Regeln definiert sind (Länge, Platzierung, Frame, Schmelztemperaturbereich), bestimmt. Die Länge der verwendeten Probes für die SNP-Detektion beträgt 60 nt. Aktuelle Methoden verwenden hier kürzere Oligos mit einer End-Punkt-Detektion für die SNP-Diskriminierung. Lange Oligos besitzen eine höhere Reproduzierbarkeit und Sensitivität, wobei die Spezifität nicht gut genug für die SNP-Detektion ist. In dieser Arbeit wird gezeigt, dass eine SNP-Diskriminierung mit langen Oligos mittels Real-Time-Analyse möglich ist.The goal of this master's thesis was to develop a real-time microarray-based SNP application. The SNP discrimination is based on the real-time analysis, hybridization kinetics and melting analysis, of DNA attached to the microarray and DNA in solution. One of the major challenges of this real-time approach is the management of the huge amount of data generated by the measurements: the measurement data of approx. 1100 spots is loaded into a self-designed MATLAB® program which sorts, normalizes and fits the data. A subsequent graphical output automatically generates diagrams of the kinetics and the melting process and views the fitting parameters in edited form, thus rendering the examination of the results easier and quicker. For the probe design a MATLAB® program was developed which automatically designs SNP microarray probes based on the sequenced gene sections from the biological material. In order to accomplish this, the SNP locations from the sequences of the two species are identified. Then the potential probe sequence variants are determined by a set of defined parameters (length, placement frames, melting temperature range). The length of the used probes for the SNP detection was 60 nt. State-of-the-art methods use shorter oligos with endpoint detection for SNP discrimination. Long oligos are known to have higher reproducibility and higher sensitivity, but the specificity is not good enough for SNP detection. Within this work it is shown that it is possible to do SNP discrimination with long oligos using real time analysis methods.author: Michael Martin PielerZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Masterarb., 2012(VLID)103587

A cell culture-derived whole virus influenza A vaccine based on magnetic sulfated cellulose particles confers protection in mice against lethal influenza A virus infection.

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening studies requiring fast purification protocols

Irreversible degradation of Nb3_3Sn Rutherford cables due to transverse compressive stress at room temperature

In the framework of the Future Circular Collider design study for a 100 TeV circular collider, 16 T superconducting bending magnets based on Nb$_3$Sn technology are being developed. A pre-stress on the conductor during magnet assembly at room temperature (RT) is needed to counteract the Lorentz forces during operation. The superconducting properties of the brittle Nb$_3$Sn superconductor are strain sensitive and excessive pre-stress leads to an irreversible degradation of the superconductor. In order to determine the level of acceptable pre-stress during the magnet assembly process, reacted and impregnated Nb$_3$Sn cables were exposed to increasing transverse compressive stress up to a maximum stress level of 200 MPa at RT. After each stress cycle, the critical current of the cable specimens were characterized at 4.3 K in the FRESCA cable test station. No significant critical current degradation was observed up to 150 MPa, followed by degradation less than 4% after a nominal stress of 175 MPa. A dramatic permanent critical current degradation occurred after applying a nominal stress of 200 MPa. A comprehensive post analysis consisting of non-destructive micro-tomography followed by microscopic characterization of metallographic cable cross sections was carried out after the critical current test to reveal cracks in the Nb$_3$Sn sub-elements of the loaded specimen

Kzf1 - a novel KRAB zinc finger protein encoding gene expressed during rat spermatogenesis.

Two novel KRAB (Krüppel associated box) type zinc finger protein encoding cDNAs, named Kzf1 and Kzf2 (Kzf for KRAB zinc finger), were identified by screening of a rat embryonic brain cDNA library with a human ZNF91 KRAB probe. Kzf1 and Kzf2 encode proteins with an amino-terminal KRAB domain and a carboxy-terminal zinc finger cluster containing 9 and 13 zinc finger units, respectively. While Kzf2 appears to be ubiquitously expressed, Kzf1 is preferentially expressed in the testis. Within the testis, Kzf1 mRNA is restricted to germ cells. The Kzf1 protein exhibits DNA binding activity and its KRAB domain can function as a repressor module in transcription. Using somatic cell hybrid analysis, the Kzf1 gene was mapped to chromosome 6.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe