Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.
Present regional practices for performing a conventional offshore structural analysis erroneously considers strengthening proposed for any additional loads on existing structures to also support the already existing loads which mainly is due to the limitations of the linear analysis approach presently adopted. This method could lead to false perception of code compliance exposing strengthened structures to a higher failure risk. The paper introduces a more accurate approach for the assessment of the structure and the evaluation of the effectiveness of strengthening introduced at different stages through the structure’s lifetime. Through the course of this study, a comparison was carried out between the conventional linear strength analysis vis-a-vis the non-linear finite element analysis technique for a typical offsore structural Bridge. The work involved full simulation of a bridge supported by two offshore structures using the conventional method and assessing the results. Subsequently an integrated finite element model, with critical locations represented through 4-node shell elements was generated. A non-linear finite element analysis was adopted where stage-wise strengthening and load history was simulated. The structural assessment using the non-linear finite element analysis technique has demonstrated that the bridge did not comply with the structural strength requirements of ISO 19902 international standards in as-is condition. This was in contradiction to the conclusion and outcome of the previous design reports. The previous strengthening design had adopted the conventional linear design methods, ignoring the realistic effects of loading and strengthening stages throughout the history of the structure. Ignoring these effects had misled to the perception of effectiveness of the new combined sections, whereas accurate representation had demonstrated that only the new additional loading is distributed to the new combined section and the existing loading is resisted by original sections only. This behavior is applicable if the installation techniques did not include jacking systems to relieve the loading prior to strengthening. The results and conclusions of the study have given an insight to the limitation of the conventional linear structural analysis methods in capturing the accurate structural behavior. It also provided a demonstration of how an incompatible type of assessment may lead to incorrect perception of compliance.
BACKGROUND: Current treatment guidelines recommend tyrosine kinase inhibitors (TKIs) as first-line and second-line treatment options for patients with chronic myeloid leukemia (CML). 1,2 However, approximately 20% of CML patients fail to respond to the first- and second-generation TKIs, and evidence-based guidelines for late-line treatment options after failure/intolerance to TKIs are limited. 3,4 Therefore, this study sought to describe the clinical characteristics and real-world treatment patterns of CML patients who received 2 prior lines of TKI therapy. METHODS: A retrospective cohort design was utilized to identify adult CML patients who initiated a new line of therapy (LOT) after 2 prior lines of TKI therapy between 4/2013 and 12/2020 in the Florida Cancer Specialists network's electronic health records (EHR) database. The index LOT date was the start date of a new CML treatment regimen in LOT≥3 after 2 prior lines of TKI therapy. Patients were followed until 1 of the following occurred: death, loss to follow-up (last structured activity in EHR), or end of study period (3/31/2021). Demographic and clinical characteristics were captured during the baseline period starting 3 months prior to the initial CML diagnosis until the index LOT date and included age, gender, race, Eastern Cooperative Oncology Group performance status (ECOG PS) and comorbidity burden. Treatment patterns of all available LOTs from initial CML diagnosis were investigated and the following metrics were captured: regimen details of systemic CML therapies, duration of LOT, treatment-free interval. This study was descriptive in nature and no inferential statistics were computed. RESULTS: A total of 157 patients who initiated a new CML treatment regimen in LOT≥3 after 2 previous lines of TKI therapy were included in this study. The median age of the study sample at initial CML diagnosis was 63.8 years, 57.3% of the sample were male, and 50.3% were White. At index LOT, 46.5% of the sample had an ECOG PS score of 0-1, 5.1% had ECOG PS score of 2-4, and 48.4% had an unknown ECOG PS score. The majority of the sample initiated the index LOT in LOT 3 (n=151; 96.2%) vs in LOT 4 (n=6; 3.8%). The median duration of the index LOT was 6.6 months with median follow-up from index LOT being 23.0 months. After 2 prior lines of TKI therapy, 93.0% of the sample continued on TKIs only, 1.3% initiated a non-TKI CML drug, and 5.7% received a combination of CML drugs. Dasatinib (31.2%) and imatinib (30.6%) were the most commonly used TKIs in the index LOT. There was significant variability in the treatment sequences across LOTs, however, 50.3% of the sample restarted the same TKI in their index LOT which had been used in a prior LOT. A total of 65 patients (41.4%) initiated a new LOT after the index LOT, and the treatment-free interval following the index LOT was 1.8 months, on average. CONCLUSIONS: The majority of the CML patients who received 2 prior lines of TKI therapy continue to cycle through additional LOTs containing TKIs, and half of the patients restart the same TKI used in a prior LOT. The findings of this study highlight the need for better novel therapies that can fill the treatment gap for CML patients who are refractory or intolerant to TKIs. REFERENCES: 1. Deininger MW, Shah NP, Altman JK, et al. Chronic Myeloid Leukemia, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2020;18(10):1385-1415. 2. Hochhaus A, Saussele S, Rosti G, et al. Chronic myeloid leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2017;28(suppl 4):iv41-iv51. 3. Perrotti D, Jamieson C, Goldman J, et al. Chronic myeloid leukemia: mechanisms of blastic transformation. J Clin Invest. 2010;120(7):2254-2264. 4. Cortes J, Lang F. Third-line therapy for chronic myeloid leukemia: current status and future directions. J Hematol Oncol. 2021;14(1):44. Disclosures Patel: Novartis Pharmaceuticals Corporation: Research Funding. Raju: Xcenda LLC: Current Employment, Other: Xcenda LLC received research funding from Novartis Pharmaceuticals Corporation. Coutinho: Xcenda LLC: Current Employment, Other: Xcenda LLC received research funding from Novartis Pharmaceuticals Corporation. Maegawa: Novartis Pharmaceuticals Corporation: Current Employment, Current equity holder in publicly-traded company. Lunacsek: Xcenda LLC: Current Employment, Other: Xcenda LLC received research funding from Novartis Pharmaceuticals Corporation. Deyoung: Xcenda LLC: Current Employment, Other: Xcenda LLC received research funding from Novartis Pharmaceuticals Corporation. Iorga: Novartis Pharmaceuticals Corporation: Current Employment, Current equity holder in publicly-traded company. Cao: Novartis Pharmaceuticals Corporation: Current Employment, Current equity holder in publicly-traded company.
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