Development of an N-1 perfusion medium to intensify seed train operation

Seed train expansion of cells before the final production step is often time-consuming and a major source of process variability. For the intensification of seed train operations there are several opportunities discussed across the biopharma industry today. One of the possibilities is to operate N-1 bioreactors in perfusion mode to shorten timelines and improve bioreactor utilization. In this work, we investigated the influence of using an expansion medium especially designed for N-1 perfusion to gain optimal results in the whole manufacturing campaign. For screening and designing an N-1 perfusion expansion medium, a scale down model which represents a typical production campaign, including the seed train, was established. Expansion medium prototypes were combined with different production media in the final production step, e.g. EX-CELL® Advanced HD Perfusion as medium designed for high-density perfusion, and Cellvento® 4CHO Medium and 4FEED as exemplary fed-batch process. After determining a suitable expansion medium formulation, the prototype was evaluated for solubility and streamlined to ensure a simple hydration and robust supply chain. Afterwards, results were confirmed using a simulated manufacturing process using benchtop bioreactor systems, showing that the positive impact of the expansion medium on the final yield is present both when using perfusion or fed-batch as final production stage. Our results indicate that using the right companion medium in seed train expansion - specifically designed for the purpose - can prepare the cells optimally for the final N-stage and increase productivity while using low CSPRs. Combining these findings with the application of a perfused N-1 step in the manufacturing campaign leads to a great opportunity for the intensification of the whole upstream process

Upstream process intensification using frozen high cell density intermediates

Typical seed train operations start by thawing of a single vial followed by several expansion steps. Reaching sufficient absolute cell numbers for production bioreactor inoculation is time-consuming and reduces plant flexibility. Besides long ramp up times, open cell culture operations are a major source of process variability. High cell density cryopreservation (HCDC) is a method of freezing cells in bags instead of vials and at higher cell densities. This offers the advantage of decoupling expansion and production: both steps can be separated in space and time. Room classification could be decreased due to fully closed processing and reproducibility increased due to a reduction of manual handling steps. Furthermore, these frozen seed train intermediates allow global distribution from a central expansion facility to decentralized global production facilities. Besides from advantages in production, these HCDC bags can be used in process development to ensure equal starting points in experimental setups. In this study, we developed a single-use bag assembly that supports closed filling, freezing, thawing, and inoculation. Before using the bag application, relevant parameters for this process from filling to inoculation were evaluated in vials with different cell lines. We found that the DMSO concentration for optimal freezing must not be higher than 7,5%. Furthermore, direct freezing at -80 °C instead of using a controlled rate freezing method is possible. Maximum concentration of DMSO in cell cultures should not be higher than 0,5 % when cryopreserved cells in bags are used for inoculation. For the idea of seed train intensification, we tested increasing freezing cell densities from 10 to 100 million cells/mL showing comparable growth. Functionality test of this HCDC method in comparison to vials was demonstrated in 4,2 L bioreactors simulating a manufacturing process. Applicability of this cryopreservation technology has been demonstrated using different bioreactors, perfusion systems, and various CHO cell lines

HAWC and Fermi-LAT detection of extended emission from the unidentified source 2HWC J2006+341

The discovery of the TeV point source 2HWC J2006+341 was reported in the second HAWC gamma-ray catalog. We present a follow-up study of this source here. The TeV emission is best described by an extended source with a soft spectrum. At GeV energies, an extended source is significantly detected in Fermi-LAT data. The matching locations, sizes, and spectra suggest that both gamma-ray detections correspond to the same source. Different scenarios for the origin of the emission are considered and we rule out an association to the pulsar PSR J2004+3429 due to extreme energetics required, if located at a distance of 10.8 kpc.Universidad de Costa Rica/[112-B9-171]/UCR/Costa RicaUniversidad de Costa Rica/[112-B6-509]/UCR/Costa RicaUniversidad de Costa Rica/[829-B5-198]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Físic

The High-Altitude water cherenkov (HAWC) observatory in México: The primary detector

The High-Altitude Water Cherenkov (HAWC) observatory is a second-generation continuously operated, wide field-of-view, TeV gamma-ray observatory. The HAWC observatory and its analysis techniques build on experience of the Milagro experiment in using ground-based water Cherenkov detectors for gamma-ray astronomy. HAWC is located on the Sierra Negra volcano in México at an elevation of 4100 meters above sea level. The completed HAWC observatory principal detector (HAWC) consists of 300 closely spaced water Cherenkov detectors, each equipped with four photomultiplier tubes to provide timing and charge information to reconstruct the extensive air shower energy and arrival direction. The HAWC observatory has been optimized to observe transient and steady emission from sources of gamma rays within an energy range from several hundred GeV to several hundred TeV. However, most of the air showers detected are initiated by cosmic rays, allowing studies of cosmic rays also to be performed. This paper describes the characteristics of the HAWC main array and its hardware.UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Físic

Combined dark matter searches towards dwarf spheroidal galaxies with Fermi-LAT, HAWC, H.E.S.S., MAGIC, and VERITAS

Cosmological and astrophysical observations suggest that 85\% of the total matter of the Universe is made of Dark Matter (DM). However, its nature remains one of the most challenging and fundamental open questions of particle physics. Assuming particle DM, this exotic form of matter cannot consist of Standard Model (SM) particles. Many models have been developed to attempt unraveling the nature of DM such as Weakly Interacting Massive Particles (WIMPs), the most favored particle candidates. WIMP annihilations and decay could produce SM particles which in turn hadronize and decay to give SM secondaries such as high energy $\gamma$ rays. In the framework of indirect DM search, observations of promising targets are used to search for signatures of DM annihilation. Among these, the dwarf spheroidal galaxies (dSphs) are commonly favored owing to their expected high DM content and negligible astrophysical background. In this work, we present the very first combination of 20 dSph observations, performed by the Fermi-LAT, HAWC, H.E.S.S., MAGIC, and VERITAS collaborations in order to maximize the sensitivity of DM searches and improve the current results. We use a joint maximum likelihood approach combining each experiment's individual analysis to derive more constraining upper limits on the WIMP DM self-annihilation cross-section as a function of DM particle mass. We present new DM constraints over the widest mass range ever reported, extending from 5 GeV to 100 TeV thanks to the combination of these five different $\gamma$-ray instruments