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

Purification of cell culture-derived influenza virus via continuous chromatography

In vaccine production downstream processing often constitutes a bottleneck in terms of process productivity and economy. One way to design more efficient purification trains could be the implementation of continuous chromatographic methods. The aim of this study was the purification of cell culture-derived influenza virus using continuous chromatography. Therefore, two chromatographic modes, flow through with CaptoCore (CC) beads and bind and elute with anion exchange (AEX) monoliths, were characterized for their ability to separate the virus from contaminating host cell protein and DNA. The starting material for the CC was treated with nuclease to decrease the DNA content and fragment size. Further, regeneration conditions for the chromatographic media, a prerequisite for successful continuous implementation, were identified and verified in sequential batch experiments. Simulated moving bed chromatography (SMB) was performed in an open loop configuration using constant switching times. In case of the CC material, two columns were located in the separation zones and two additional columns were regenerated and equilibrated in detached zones. For the AEX runs, on the other hand, monoliths were used in a three zone configuration with detached high salt zone for regeneration. Results in batch chromatography (BC) and SMB showed similar product yields in the range 60 to 100%. Contaminant depletion was \u3e98% DNA and \u3e58% protein for the AEX monoliths. Both the CC SMB and the BC resulted in comparable impurity levels (33.2 µg protein and 25.6 ng DNA per estimated 15 µg HA) but for BC a higher product yield (89% vs 72%) was achieved. In addition, the virus dilution during the flow through chromatography could be reduced in the cyclic steady state of the SMB by a factor of 1.8. Overall, the separation performance of the BC has been successfully transferred to the continuous process

A new simplified clarification approach for lentiviral vectors using diatomaceous earth improves throughput and safe handling

Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process

Optical/IR from ground

Optical/infrared (O/IR) astronomy in the 1990's is reviewed. The following subject areas are included: research environment; science opportunities; technical development of the 1980's and opportunities for the 1990's; and ground-based O/IR astronomy outside the U.S. Recommendations are presented for: (1) large scale programs (Priority 1: a coordinated program for large O/IR telescopes); (2) medium scale programs (Priority 1: a coordinated program for high angular resolution; Priority 2: a new generation of 4-m class telescopes); (3) small scale programs (Priority 1: near-IR and optical all-sky surveys; Priority 2: a National Astrometric Facility); and (4) infrastructure issues (develop, purchase, and distribute optical CCDs and infrared arrays; a program to support large optics technology; a new generation of large filled aperture telescopes; a program to archive and disseminate astronomical databases; and a program for training new instrumentalists

Continuous purification of cell culture-derived influenza A virus particles through pseudo- affinity membrane chromatography

Continuous manufacturing is a relevant trend in biopharmaceutical production to reduce the process footprint and to improve the process economy. Vaccines against world-spread diseases, such as influenza, should benefit in particular from such an approach, given the increasing demand for seasonal vaccines and the need for a fast response in case of a pandemic outbreak. Upstream processing of viral vaccines has seen important progress in continuous production of viral vaccines [1], which further supports the development of hybrid or fully continuous flow-schemes for downstream processing. Please click Additional Files below to see the full abstract

GEANN: Scalable Graph Augmentations for Multi-Horizon Time Series Forecasting

Encoder-decoder deep neural networks have been increasingly studied for multi-horizon time series forecasting, especially in real-world applications. However, to forecast accurately, these sophisticated models typically rely on a large number of time series examples with substantial history. A rapidly growing topic of interest is forecasting time series which lack sufficient historical data -- often referred to as the ``cold start'' problem. In this paper, we introduce a novel yet simple method to address this problem by leveraging graph neural networks (GNNs) as a data augmentation for enhancing the encoder used by such forecasters. These GNN-based features can capture complex inter-series relationships, and their generation process can be optimized end-to-end with the forecasting task. We show that our architecture can use either data-driven or domain knowledge-defined graphs, scaling to incorporate information from multiple very large graphs with millions of nodes. In our target application of demand forecasting for a large e-commerce retailer, we demonstrate on both a small dataset of 100K products and a large dataset with over 2 million products that our method improves overall performance over competitive baseline models. More importantly, we show that it brings substantially more gains to ``cold start'' products such as those newly launched or recently out-of-stock

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context

The Ever Changing Circumstellar Nebula Around UW Centauri

We present new images of the reflection nebula surrounding the R Coronae Borealis Star, UW Cen. This nebula, first detected in 1990, has changed its appearance significantly. At the estimated distance of UW Cen, this nebula is approximately 0.6 ly in radius so the nebula cannot have physically altered in only 8 years. Instead, the morphology of the nebula appears to change as different parts are illuminated by light from the central star modulated by shifting thick dust clouds near its surface. These dust clouds form and dissipate at irregular intervals causing the well-known declines in the R Coronae Borealis (RCB) stars. In this way, the central star acts like a lighthouse shining through holes in the dust clouds and lighting up different portions of the nebula. The existence of this nebula provides clues to the evolutionary history of RCB stars possibly linking them to the Planetary Nebulae and the final helium shell flash stars.Comment: To be published in ApJ Letters. 5 pages, 3 figures (2 in color