Controlling tryptophan oxidation through medium/feed modifications and potential MOA unveiled by transcriptomics analysis

Oxidation of monoclonal antibodies (mAbs) is one of the major product quality issues with potential impacts on product activity and immunogenicity. Tryptophan oxidation (Trp-ox) leads to addition of one or two oxygen atoms to the indole ring of its side chain and other subsequent degradation end products. It was reported that Trp-ox in the complementarity-determining region of a mAb led to a progressive loss of antigen binding and biological activity 1. Trp-ox was also reported to cause color changes in near UV−visible light-irradiated and heat-stressed monoclonal antibody (mAb) drug product in liquid formulation 2. In addition, Trp-ox was observed under real-time storage and elevated temperature conditions 3. Recently, we observed that modifying the concentrations of copper, manganese, tryptophan, and cysteine in cell culture media/feed had a significant impact on Trp-ox levels of two mAbs in development produced in Chinese hamster ovary (CHO) cells 4. We have demonstrated that Trp-ox level can be effectively controlled while maintaining productivity and overall suitable product quality profiles. In this presentation we will summarize those findings and the results from the systematic studies that enabled us to control the Trp-ox levels at both the shake flask and benchtop bioreactor scales. Moreover, we will describe new studies that aimed to understand the potential mechanism of action (MOA) of those components on controlling Trp-ox levels. The advent of NGS technologies and the availability of CHO reference genomes have enabled the systematic analysis of CHO biology and its capacity for recombinant protein production 5, 6. Here we applied transcriptomic analysis using RNA-Seq to explore the underlying mechanisms of cell culture’s impact on Trp-ox. Cell samples from fed-batch bioreactors cultured with control or modified media/feed were harvested and subjected to RNA-Seq analysis. The results showed that cell culture conditions had little impact on the expression of the mAb transgenes (LC and HC), nor genes related to glycosylation, which is consistent with the previous findings on mAb productivity and glycosylation profile 4. However, cell culture conditions did significantly alter the expression of multiple genes (fold change ≥1.5, p-value £0.05). Specific subsets of genes involved in control of oxidative stress and metabolism of copper, manganese, tryptophan cysteine will be discussed in detail. The analyses will focus on genes engaged in scavenging of free radicals because of their known roles in oxidation chemistry and production of reactive oxygen species (ROS). We postulate that these changes in gene expression may provide molecular means to balance the copper availability and glutathione pool, which in turn might result in the observed impact on mAb quality without changing the CHO cell growth and productivity. The work presented here provide another example of how gene expression analyses can shed additional light on potential mechanisms for observed cell culture performance and specifically in this case, changes in recombinant protein product quality attributes. Such understanding could eventually lead to a biomarker-based approach for process optimizations. To the best of our knowledge, this is the first example of using transcriptomic analysis to mechanistically understand the impact of cell culture on critical quality attributes other than glycosylation. Therefore, we believe this presentation is of great interest to general biopharmaceutical community and is relevant the themes of the Conference, especially to the section “Advances in cell culture control of product quality attributes”. 1. Wei, Z. et al. Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal Chem 79, 2797-2805 (2007). 2. Li, Y., Polozova, A., Gruia, F. & Feng, J. Characterization of the degradation products of a color-changed monoclonal antibody: tryptophan-derived chromophores. Anal Chem 86, 6850-6857 (2014). 3. Hensel, M. et al. Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS. PLoS One 6, e17708 (2011). 4. Hazeltine, L.B. et al. Chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals. Biotechnol Prog 32, 178-188 (2016). 5. Kildegaard, H.F., Baycin-Hizal, D., Lewis, N.E. & Betenbaugh, M.J. The emerging CHO systems biology era: harnessing the ‘omics revolution for biotechnology. Current Opinion in Biotechnology 24, 1102-1107 (2013). 6.Richelle, A. & Lewis, N.E. Improvements in protein production in mammalian cells from targeted metabolic engineering. Current Opinion in Systems Biology 6, 1-6 (2017)

Effectiveness and safety of serial endoscopic ultrasound–guided celiac plexus block for chronic pancreatitis

Background and study aims: Endoscopic ultrasound – guided celiac plexus block (EUS-CPB) is an established treatment for pain in patients with chronic pancreatitis (CP), but the effectiveness and safety of repeated procedures are unknown. Our objective is to report our experience of repeated EUS-CPB procedures within a single patient. , Patients and methods: A prospectively maintained EUS database was retrospectively analyzed to identify patients who had undergone more than one EUS-CPB procedure over a 17-year period. The main outcome measures included number of EUS-CPB procedures for each patient, self-reported pain relief, duration of pain relief, and procedure-related adverse events. , Results: A total of 248 patients underwent more than one EUS-CPB procedure and were included in our study. Patients with known or suspected CP (N = 248) underwent a mean (SD) of 3.1 (1.6) EUS-CPB procedures. In 76 % of the patients with CP, the median (range) duration of the response to the first EUS-CPB procedure was 10 (1 – 54) weeks. Lack of pain relief after the initial EUS-CPB was associated with failure of the next EUS-CPB (OR 0.17, 95 %CI 0.06 – 0.54). Older age at first EUS-CPB and pain relief after the first EUS-CPB were significantly associated with pain relief after subsequent blocks (P = 0.026 and P = 0.002, respectively). Adverse events included peri-procedural hypoxia (n = 2) and hypotension (n = 1) and post-procedural orthostasis (n = 2) and diarrhea (n = 4). No major adverse events occurred., Conclusions: Repeated EUS-CPB procedures in a single patient appear to be safe. Response to the first EUS-CPB is associated with response to subsequent blocks

Versuche uber Immunisierung von dem Konjunktivalsack aus mit Berucksichtigung des Einflusses der Ultraviolettbestrahlung auf den Immunisierungsvorgang

1) In Vorversuchen hat der Verfasser eingehend einerseits den direkten Einfluss der Ultraviolettstrahlen auf die beabsichtigte Antigene, ihre Suspensions-bzw. Losungs-medien so wie auf die in Immunserum anwesenden Antikorper festgestellt, anderseits die lokale und allgemeine Einwirkung auf den Tierkorper der U. V. Bestrahlung des Kaninchenauges sichergestellt. Im allgemeinen hat die U. V. Bestrahlung auf lebende Bakterien verschiedener Rassen bakterizide Wirkung entfaltet. Die Wirkungskraft war bei verschiedenen Bakterienarten, in verschiedenen Suspensionsmedien und je nach der Bestrahlungsweise verschieden. Auf das Auge selbst hat die U. V. Bestrahlung, vorausgesetzt, dass sie zweck-massig durchgefuhrt wird, keinen ublen Einfluss. Die Keime des Konjunktivalsackes nehmen als Folge der Bestrahlung an ihrer Zahl ab. Als allgemeiner Einfluss auf den Tierkorper kann der Aufstieg der Korpertem-peratur nach der Bestrahlung und die Korpergewichtzunahme erwahnt werden. Uber-massiges Bestrahlen fuhrt zur Gewichtsabnahme. Im Gegenteil zu der Wirkung im immuisierten Korper verliert das direkt bestr-ahlte Immunserum an seinem Antikorpergehalt. 2) Durch Agglutinationsproben wurde der Immunisierungsvorgang verfolgt und die Moglichkeit der Immunisierung vom Konjumktivalsack aus festgestellt. Es kamen die folgenden Bakterien in Frage; B. typhi B. prodigiosus, Staphylokokken, Gonoko-kken, Pneumokokken, V. Metschnikoffi. Als Aufschwemmungsmedium der bakteriellen Antigene wurde Kochsalzlosung, Dionin-, NaHCO3-, und verdunnte HCl- losung gebraucht. Diese Immunisierungsversuche wurden teilweise mit, teilweise ohne U. V. Bestrahlung durchgefuhrt, wodurch die Tatsache festgestellt wurde, dass die Bestr-ahlung auf die Agglutininbildung einen gunstigen Einfluss ausubt. 3) Ahnlich wie mit den verschiedenen Bakterien wurden auch Immunisierungs-versuche mit Diphterietoxin angestellt. Es ist zwar gelungen im Blute der auf die in Frage stehende Weise behandelten Versuchstiere das spezifische Antitoxin nachzuweisen, aber es lag kein Beweis a

The Drosophila maternal-effect mutantcappuccino and its interactors

cappuccino (capu) is a Drosophila melanogaster gene required for establishing the dorsal-ventral and anterior-posterior axes of the developing egg and embryo. Egg chambers mutant for capu exhibit cytoplasmic streaming during mid-oogenesis that does not normally occur until late oogenesis. All known capu alleles stream at the same speed. Since capu alleles do differ in their effects on the dorsal-ventral axis, it is unlikely that premature streaming is a cause of the dorsal-ventral defects. Premature streaming may, however, cause the posterior defects. Streaming occurs at the same speed if isolated egg chambers are treated with the actin depolymerizing drug cytochalasin D, which suggests that CAPU may act in the actin cytoskeleton. A screen for proteins which physically interact with CAPU identified profilin, a regulator of the actin cytoskeleton as a probable partner of CAPU. This again suggests that CAPU acts in the actin cytoskeleton. CAPU is a member of the formin homology (FH) family of proteins. Sequence analysis of this family makes it possible to multiply align all family members throughout their carboxy-terminal halves. This makes possible better predictions of secondary structure, phylogenetic analysis, and identification of novel regions of conserved sequence. Analysis of the amino-terminal halves suggests that significant alignment is also possible in these more highly divergent regions. Members of the rho family of small GTPases have been implicated in the regulation of the actin cytoskeleton. They physically associate with members of the FH family, including CAPU. All four rho-like proteins tested associate with CAPU in the Interaction Trap system. There are also indications of genetic interactions between capu and the rho-like genes dcdc42 and drac1

Sexual Violence against Women during Conflict

abstract: The purpose of this honors thesis is to explain the varying levels of sexual violence against women across time, location and conflicts. Violence against civilians is utilized as an independent variable to measure if the level of violence of a pre-conflict environment widens the space for the exploitation of sexual violence. Women's status is used as an additional independent variable in order to measure if a pre-conflict environment that promotes gender equality moderates the presence of sexual violence as it discourages unequal power dynamics. GDP per capita and population will be used as control variables in order to include consideration of state capacity. Sexual violence will be the dependent variable. In order to statistically measure and depict the relationships between these variables, bivariate correlations and multivariate linear regressions will be utilized. The bivariate correlations showed that as civilian violence increased, sexual violence increased as well, but as women's status increased, sexual violence decreased. The linear regression models found that state actors and rebel groups yielded differing results. For state actors, the increase in women's status failed to moderate the level of sexual violence as an increase in civilian violence and women's status resulted in an increase in sexual violence. However, for rebel groups, an increase in civilian violence and women's status led to a decrease in sexual violence, thereby depicting women's status as a moderating factor. This creates a problem in identifying one or a few factors that predominately lead to an increase in sexual violence; such identification is key for the development of preventative policy

Additional file 8: Table S4. of Genomic structure and expression of the human serotonin 2A receptor gene (HTR2A) locus: identification of novel HTR2A and antisense (HTR2A-AS1) exons

Mouse Htr2a exons and coordinates. (XLSX 12 kb