From screening to process optimization: AMBR technology to speed up microbial fermentation processes

Session proposals: · Therapeutic Proteins Vaccines The development of biopharmaceuticals or biotechnological products derived from microbial fermentation is a financially risky endeavor and time consuming process, requiring technical upstream solutions which reduce timelines, increase efficiency, and raise likelihood of success. We have identified in particular the early steps of strain and process development offering best prospects to speed up the entire process significantly by using a reliable screening system. Based on the well-proven ambr® principle we designed with ambr 15 fermentation system to accelerate early stage development of microbial fermentation products. The multi-fermentation unit mimics larger scale bioreactor processes, and is suitable for screening clones, strains or growth conditions. In case studies with industrial partners using E. coli and P. pastoris, consistent and efficient control of fermentations across a variety culture conditions (e.g. feed, temperature, duration, pH) could be demonstrated. In the succeeding step of process development ambr 250 has been widely applied to speed up the 2nd critical phase of the microbial upstream process development. The larger working volume and the range of features, which this multi-parallel system offers, are superior to common benchtop fermenters. Optical density supervision, off gas analysis, fed-batch processing and advanced control capabilities allow process development for most commercial-scale upstream fermentation processes. In addition to this impressive range of features ambr250 has proven its ability to reliably increase the efficiency of fermentation process development many times through its rapid setup and cleanup, advanced control software, and automation

Bioprocess intensification for production of a Peste des petits ruminants virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, targeted by the Food and Agriculture Organization for 2030, a novel and scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in the 2L BIOSTAT® DCU-3 and the 20L BIOSTAT® Cplus STB (both from Sartorius) using Nigeria 75/1 strain. Engineering correlations (energy dissipation rate, shear stress and Kolmogorov Eddy size) were used to optimize culture conditions in the 2 L STB and to scale-up the process to the 20 L STB. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. Perfusion was evaluated in the 2 L STB (equipped with internal spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP3, Sartorius). Process scalability was validated in the 20 L STB. Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates similar to serum-containing cultures (0.03 h-1). The new in situ cell detachment method was successfully implemented, with yields above 80%. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the new in situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. Infectious PPRV titers increase over culture time in both 2 L and 20 L STBs, reaching maximum values of 4.5-4.9x106 TCID50/mL at day 4-5 post-infection. The potential of depth filtration for PPRV clarification was confirmed; comparable PPRV recovery yields after clarification (85-90%) were obtained in both STBs. Overall, the novel and scalable vaccine production process herein proposed has the potential to assist the upcoming PPR Global Eradication Program (PPR GEP), to which iBET already contributes as partner in the PPR Global Research and Experts Network (PPR GREN), and thus support the One Health concept. Acknowledgments: This work was supported by Sartorius Stedim Biotech GmbH (Germany)

Upstream microbial process characterization with single-use bioreactors from 15 mL to 50L

Developing biological and industrial molecules derived from microbial fermentation relies upon performant bioreactors to allow a rapid scale up to commercial batches. For this it is relevant to minimize any possible risks while developing a process that fits the industry quality standards. The choice of a well characterized system plays an important role from R&D through to production stages. The aim of this poster is to provide evidence to demonstrate the benefits of a microbial process developed using single-use, high throughput, and scalable upstream solutions. The method chosen to showcase this consistency is based on the DECHEMA Guidelines for Engineering Characterization principles and with the Zurich University of Applied Sciences, ZHAW. DECHEMA guidelines include a set of standard conditions for bioreactor characterization. By using process development and pilot scale bioreactors like the ambr 15f, ambr 250, and BIOSTAT STR 50, it is possible to accelerate development timelines and ensure process success

Linking Personality Traits to Individual Differences in Affective Spaces

Different individuals respond differently to emotional stimuli in their environment. Therefore, to understand how emotions are represented mentally will ultimately require investigations into individual-level information. Here we tasked participants with freely arranging emotionally charged images on a computer screen according to their subjective emotional similarity (yielding a unique affective space for each participant) and subsequently sought external validity of the layout of the individuals' affective spaces through the five-factor personality model (Neuroticism, Extraversion, Openness to Experience, Agreeableness, Conscientiousness) assessed via the NEO Five-Factor Inventory. Applying agglomerative hierarchical clustering to the group-level affective space revealed a set of underlying affective clusters whose within-cluster dissimilarity, per individual, was then correlated with individuals' personality scores. These cluster-based analyses predominantly revealed that the dispersion of the negative cluster showed a positive relationship with Neuroticism and a negative relationship with Conscientiousness, a finding that would be predicted by prior work. Such results demonstrate the non-spurious structure of individualized emotion information revealed by data-driven analyses of a behavioral task (and validated by incorporating psychological measures of personality) and corroborate prior knowledge of the interaction between affect and personality. Future investigations can similarly combine hypothesis- and data-driven methods to extend such findings, potentially yielding new perspectives on underlying cognitive processes, disease susceptibility, or even diagnostic/prognostic markers for mental disorders involving emotion dysregulation

Local and global effects of sedation in resting-state fMRI: a randomized, placebo-controlled comparison between etifoxine and alprazolam

TSPO ligands are promising alternatives to benzodiazepines in the treatment of anxiety, as they display less pronounced side effects such as sedation, cognitive impairment, tolerance development and abuse potential. In a randomized double-blind repeated-measures study we compare a benzodiazepine (alprazolam) to a TSPO ligand (etifoxine) by assessing side effects and acquiring resting-state fMRI data from 34 healthy participants after 5 days of taking alprazolam, etifoxine or a placebo. To study the effects of the pharmacological interventions in fMRI in detail and across different scales, we combine in our study complementary analysis strategies related to whole-brain functional network connectivity, local connectivity analysis expressed in regional homogeneity, fluctuations in low-frequency BOLD amplitudes and coherency of independent resting-state networks. Participants reported considerable adverse effects such as fatigue, sleepiness and concentration impairments, related to the administration of alprazolam compared to placebo. In resting-state fMRI we found a significant decrease in functional connection density, network efficiency and a decrease in the networks rich-club coefficient related to alprazolam. While observing a general decrease in regional homogeneity in high-level brain networks in the alprazolam condition, we simultaneously could detect an increase in regional homogeneity and resting-state network coherence in low-level sensory regions. Further we found a general increase in the low-frequency compartment of the BOLD signal. In the etifoxine condition, participants did not report any significant side effects compared to the placebo, and we did not observe any corresponding modulations in our fMRI metrics. Our results are consistent with the idea that sedation globally disconnects low-level functional networks, but simultaneously increases their within-connectivity. Further, our results point towards the potential of TSPO ligands in the treatment of anxiety and depression

Short-term effects of etifoxine on human gut microbiome in healthy men

BackgroundNeurosteroids have recently gained in interest as a treatment strategy for affective disorders. Etifoxine is known for its dual mode of action, one of which is to stimulate endogenous neurosteroid synthesis. The gut microbiome has been studied in affective disorders, but it has not been investigated in the context of human etifoxine or neurosteroid interventions.MethodsWe performed a crossover study with 36 healthy male volunteers who received etifoxine versus alprazolam and placebo in a balanced Williams design. Participants were randomized into six sequences and went through three 5-day treatments followed by wash-out phases of 9 days. Bacterial compositions in stool samples were determined by high-throughput 16S rRNA amplicon sequencing.ResultsGut microbiome analyses revealed no relevant effects between treatments with respect to alpha and beta diversity. Differential abundance analyses yielded etifoxine treatment as the only effect related to changes in microbial features with reductions of Faecalibacterium duncaniae, Roseburia hominis and Lactobacillus rogosae (i.e., Bacteroides galacturonicus).ConclusionHere we report on the first human investigation of the gut microbiome with short-term etifoxine intervention. Differences in diversity and compositional structure of the microbiome were more likely due to between- subject effects rather than medication. However, five-day treatment with etifoxine reduced the abundance of a few bacterial species. These species are currently seen as beneficial components of a healthy intestinal microbiome. This reduction in abundances may be related to elevated endogenous neurosteroids

A database of naturally occurring human urinary peptides and proteins for use in clinical applications

Owing to its availability, ease of collection and correlation with (patho-) physiology, urine is an attractive source for clinical proteomics. However, the lack of comparable datasets from large cohorts has greatly hindered development in this field. Here we report the establishment of a high resolution proteome database of naturally occurring human urinary peptides and proteins - ranging from 800-17,000 Da - from over 3,600 individual samples using capillary electrophoresis coupled to mass spectrometry, yielding an average of 1,500 peptides per sample. All processed data were deposited in an SQL database, currently containing 5,010 relevant unique urinary peptides that serve as classifiers for diagnosis and monitoring of diseases, including kidney and vascular diseases. Of these, 352 have been sequenced to date. To demonstrate the applicability of this database, two examples of disease diagnosis were provided: For renal damage diagnosis, patients with a specific renal disease were identified with high specificity and sensitivity in a blinded cohort of 131 individuals. We further show definition of biomarkers specific for immunosuppression and complications after transplantation (Kaposi's sarcoma). Due to its high information content, this database will be a powerful tool for the validation of biomarkers for both renal and non-renal diseases

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days