Soluble papain to digest monoclonal antibodies; time and cost-effective method to obtain Fab fragment

Antigen binding fragments (Fabs) used in research (e.g., antibody mimetics, antibody-drug conjugate, bispecific antibodies) are frequently obtained by enzymatic digestion of monoclonal antibodies using immobilised papain. Despite obtaining pure Fab, using immobilised papain to digest IgG has limitations, most notably slow digestion time (more than 8 h), high cost and limited scalability. Here we report a time and cost-effective method to produce pure, active and stable Fab using soluble papain. Large laboratory scale digestion of an antibody (100 mg) was achieved using soluble papain with a digestion time of 30 min and isolated yields of 55–60%. The obtained Fabs displayed similar binding activity as Fabs prepared via immobilised papain digestion. Site-specific conjugation between Fabs and polyethylene glycol (PEG) was carried out to obtain antibody mimetics FpF (Fab-PEG-Fab) indicating that the native disulphide bond had been preserved. Surface-plasmon resonance (SPR) of prepared FpFs showed that binding activity towards the intended antigen was maintained. We anticipate that this work will provide a fast and less costly method for researchers to produce antibody fragments at large scale from whole IgG suitable for use in research

Advanced Therapy Medicinal Products for Age-Related Macular Degeneration; Scaffold Fabrication and Delivery Methods

Retinal degenerative diseases such as age-related macular degeneration (AMD) represent a leading cause of blindness, resulting in permanent damage to retinal cells that are essential for maintaining normal vision. Around 12% of people over the age of 65 have some form of retinal degenerative disease. Whilst antibody-based drugs have revolutionised treatment of neovascular AMD, they are only effective at an early stage and cannot prevent eventual progression or allow recovery of previously lost vision. Hence, there is a clear unmet need to find innovative treatment strategies to develop a long-term cure. The replacement of damaged retinal cells is thought to be the best therapeutic strategy for the treatment of patients with retinal degeneration. Advanced therapy medicinal products (ATMPs) are a group of innovative and complex biological products including cell therapy medicinal products, gene therapy medicinal products, and tissue engineered products. Development of ATMPs for the treatment of retinal degeneration diseases has become a fast-growing field of research because it offers the potential to replace damaged retinal cells for long-term treatment of AMD. While gene therapy has shown encouraging results, its effectiveness for treatment of retinal disease may be hampered by the body’s response and problems associated with inflammation in the eye. In this mini-review, we focus on describing ATMP approaches including cell- and gene-based therapies for treatment of AMD along with their applications. We also aim to provide a brief overview of biological substitutes, also known as scaffolds, that can be used for delivery of cells to the target tissue and describe biomechanical properties required for optimal delivery. We describe different fabrication methods for preparing cell-scaffolds and explain how the use of artificial intelligence (AI) can aid with the process. We predict that combining AI with 3D bioprinting for 3D cell-scaffold fabrication could potentially revolutionise retinal tissue engineering and open up new opportunities for developing innovative platforms to deliver therapeutic agents to the target tissues

Cosmetic Breast Implants and the Risk of Suicide: A Systematic Review and Meta-Analysis

Objective: Having cosmetic breast implants increases a woman's chance of suicide, which is now a global challenge. This systematic review evaluated the possible risk of suicide among women who undergo cosmetic breast implants. Method: This meta-analysis was done based on Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA). In the current systematic review and meta-analysis, we systematically searched for all articles written in both English or Persian that estimated the prevalence of suicidal ideation in women who had cosmetic breast implants. We systematically searched different databases, including MEDLINE (PubMed), Web of Science, Embase, Cochrane, Library ProQuest, Scopus, and Google Scholar, from inception to March 2021. There was also a search for references. Suicidal ideation, a suicide plan, or suicide attempts were the outcomes. In order to determine the total pooled prevalence of suicidal ideation, we utilized a random-effects model. To examine the risks of bias in each study, we applied the Joanna Briggs Institute Critical Appraisal method. Results: We identified 218 citations in our initial search. After omitting duplicated citations and excluding irrelevant studies according to the title and abstract selection, 42 studies were chosen for the full text analysis. Finally, 11 research, examining a total of 324,332 women were incorporated into the systematic review and critical appraisal assessment. Eight of these studies were found to be eligible for meta-analysis. The frequency of suicide in women with cosmetic breast implant was 0.2% (95% CI: 0.1% to 0.4%; P < 0.001) (Q-value: 168.143, I2:95.83). Most of the included studies had moderate quality. Conclusion: There might be a correlation between cosmetic breast implants and suicide risk, which could be stronger in the presence of a history of mental illnesses. The evidence about the possible effects of breast implants on the risk of suicide is still inconclusive, and there is a need for future well-designed studies on this topic

HYSSOP and POLIUM could help to prevent COVID-19 in high-risk population: The results of a parallel randomized placebo-controlled field trial

243-253This study was conducted to evaluate the effect of HYSSOP (composed of Hyssopus officinalis L., Echium amoenum Fisch & C. A. Mey and Glycyrrhiza glabra L.) and POLIUM (contained Teucrium polium L., Cuscuta epithymum Murr and Cichorium intybus L.) combined distilled herbal medicines compared to placebo in the prevention of COVID-19. This is a double-blind parallel placebo-controlled field trial conducted on 751 asymptomatic individuals whose one of the family members recently had a positive RT-PCR test for COVID-19. They were divided into three groups including POLIUM, HYSSOP and placebo using random blocks with a 1:1:1 allocation ratio. Participants received daily 5 cc (under 12 years) or 10 cc (over 12 years) of allocated oral medications for 20 days. The primary outcome was the frequency of positive RT-PCR test among participants who became symptomatic. The mean age of participants was 36.6. Nineteen participants get infected by COVID-19 during the intervention; fifteen of them belonged to the placebo and four to the POLIUM group. Fisher's exact test indicated significant differences between HYSSOP and placebo (p<0.001) as well as POLIUM and placebo (p=0.009) groups in terms of COVID-19 confirmed by PCR tests. Cox regression model adjusted for confounders illustrated that the hazard of getting infection by COVID-19 in POLIUM and HYSSOP groups decreased by 66% (OR (95% CI): 0.34 (0.12 to 0.94); p=0.038) and 93% (OR (95% CI): 0.07 (0.01to 0.56); p=0.012) respectively, compared to placebo .Oral administration of HYSSOP and POLIUM with the other supportive health care could decrease the risk of getting COVID-19

Circulating power: humanitarian logistics, militarism and the United Arab Emirates

While critical authors have interrogated the roots of business logistics, this paper extends the analysis and contributes to a larger critique of the cohering field of Humanitarian Logistics (HL), noting the overlap in the logistical cartographies of militarism and humanitarianism. The focus is on the UAE’s expanding logistics space into the Horn of Africa and the production of specialised HL zones like Dubai International Humanitarian City (DIHC). The article makes three core arguments. First, a logistics lens enables us to expand the study of aid beyond immediate conflict zones, into more distant spaces often constructed as ‘stable’. Second, the placement of logistics at the core of aid delivery has been a key mechanism for inserting market imperatives into humanitarian activities. Third, this gives countries outside the advanced core, such as the UAE, power to leverage and expand their logistics space for multiple purposes, in war making, aid, and commercial activities

Infrastructures of empire: towards a critical geopolitics of media and information studies

The Arab Uprisings of 2011 can be seen as a turning point for media and information studies scholars, many of whom newly discovered the region as a site for theories of digital media and social transformation. This work has argued that digital media technologies fuel or transform political change through new networked publics, new forms of connective action cultivating liberal democratic values. These works have, surprisingly, little to say about the United States and other Western colonial powers’ legacy of occupation, ongoing violence and strategic interests in the region. It is as if the Arab Spring was a vindication for the universal appeal of Western liberal democracy delivered through the gift of the Internet, social media as manifestation of the ‘technologies of freedom’ long promised by Cold War. We propose an alternate trajectory in terms of reorienting discussions of media and information infrastructures as embedded within the resurgence of idealized liberal democratic norms in the wake of the end of the Cold War. We look at the demise of the media and empire debates and ‘the rise of the BRICS’ (Brazil, Russia, India, China, South Africa) as modes of intra-imperial competition that complicate earlier Eurocentric narratives media and empire. We then outline the individual contributions for the special collection of essays

Disulfide-bridging PEGylation of antibody fragments

Monoclonal antibodies are routinely used in the clinic. There is also a small number of antibody fragments (e.g. Fabs) that are clinically used. In many applications where antibody binding is required to antagonise a receptor or simply to bind a ligand, there is no need for the Fe properties that are associated with effector functions. Unfortunately, without the Fe region, monovalent antibody fragments rapidly clear from the blood circulation. PEGylation is the most successful and clinically used approach to date for increasing the circulation half-life of therapeutic proteins. Disulfide bridging PEGylation is an appropriate method to site-specifically conjugate a molecule of PEG at the interchain disulfide of a Fab. Since a PEGylated Fab will be monovalent, it is not possible to exploit the cooperative binding or avidity that is associated with the bivalency of a full IgG. An objective of this PhD project was firstly to understand if a monovalent PEGylated Fab can bind effectively to its antigen. A bis-alkylation PEG reagent with a functional group at each terminus of the PEG could then be used to conjugate two Fabs, one at each end of the PEG molecule, to generate either a homo Fab-PEG-Fab or a hetero Fab-PEG-Fab* conjugate. An important objective of this PhD was to determine the structure-property correlations of a small family of PEGylated-Fabs. It was hypothesised that Fab-PEG- Fab conjugates would display comparable binding properties to the full parent IgG. Three Fabs were PEGylated in this project. Fabbeva and Fabtrast were obtained by papain digestion of bevacizumab and trastuzumab respectively. Ranibizumab is a clinically used Fab and therefore did not require digestion of a full antibody. Both Fabbeva and Fabrani bind to VEGF and Fabtrast binds to HER-2. After treament with DTT to open the interchain disulfide, each Fab underwent reaction with a Fab reagent capable of thiol specific, bis-alkylation. PEGylation was accomplished at near quantitative conversion with 1-2 equivalents with reproducible result. A single step ion-exchange purification process was used to obtain purified mono PEG-Fabs. The PEG-Fab conjugates were stable during a 3 months stability study at 4 °c with no de- PEGylation. A PEG2x2o-Fab' beva construct was also generated by conjugation of two molecules of the PEG reagent to intrachain disulfide bonds of the Fab beva- BIAcore and ELISA studies confirmed that compared with the unPEGylated Fabs, the PEGylated Fabbeva, Fabrani and Fabtrast displayed a 2 fold decrease in binding affinity I for their respective ligands. This decrease in binding affinity was much less than had been reported in the literature. PEG-Fabbeva conjugates comprising 20, 30 and 40 kDa PEG all displayed similar binding affinities. The binding affinity of the PEG2x20- Fab beva was decreased compared with mono PEG2o-Fabbeva as a result of a change in the dissociation rate constant. The homodimer Fab-PEG-Fab constructs comprising 6, 10 and 20 kDa PEG and Fabbeva, Fabrani and Fabtrast maintained their binding affinities compared with the parent IgGs. BIAcore kinetic studies showed there was greater binding affinity and slower dissociation rate for the Fabbeva-PEG-Fabbeva than the native Fabbeva. While similar binding affinity to bevacizumab was observed for the Fabbeva-PEG-Fabbeva, the dissociation rates of the the Fabbeva-PEG-Fabbeva were slower than for bevacizumab. It was also found that using a longer PEG in the Fabbeva-PEG-Fabbeva resulted in slower dissociation. The heterodimer Fabbeva-PEG2o-Fabtrast* that was produced maintained binding to VEGF and HER-2. An in vitro angiogenesis assay suggested that the Fabbeva-PEG20-Fabbeva and Fabrani-PEG20-Fabrani inhibit angiogenesis more effectively than bevacizumab. Using PEG as a linking molecule to conjugate two Fabs would appear to be a valid way to provide bivalency to the molecule, resulting in at least the functional activity expected of a full IgG. Since the PEG is a flexible coil, it may be the case that the two conjugated Fabs in the Fab-PEG-Fab homodimer are brought towards the VEGF in a manner that is more efficient than for a native IgG, resulting in a stronger binding interaction and hence enhanced functional activity. These results for Fab- PEG-Fab homodimer are encouraging and together with the results for the Fab-PEG- Fab* bring the potential to aid development of bivalent and bispecific protein-based medicines.EThOS - Electronic Theses Online ServiceGBUnited Kingdo