1,720,960 research outputs found
Investigating Ultrafiltration Membranes and Operation Modes for Improved Lentiviral Vector Processing
No full textABSTRACT The demand for lentiviral vectors (LVs) as tools for ex vivo gene therapies is ever‐increasing. Despite their promising applications, challenges in LV production remain largely due to the fragile envelope, which challenges the maintenance of vector stability. Thus, downstream processing optimization to enhance efficiency, yield, and product quality is necessary. This study investigated the influence of membrane types and filtration devices during ultrafiltration (UF). Nine different membrane materials consisting of polyethersulfone (PES), regenerated cellulose, or Hydrosart, with distinct molecular weight cutoffs, were evaluated in stirred cells, centrifugal ultrafilters, and crossflow cassettes. The evaluation was based on the ability to retain infectious LV particles and remove impurities. The analysis revealed that a reinforced 100 kDa PES and a 300 kDa Hydrosart membrane had the best overall ability to concentrate infectious LVs and remove DNA, especially when operated in a stirred cell. Challenges were seen in the nonoptimized crossflow cassette process, where infectious LV recovery was generally lower compared to other devices. We demonstrated that membrane material and filtration device have a direct impact on the efficiency of LV UF
A robust and efficient alluvial filtration method for the clarification of adeno-associated viruses from crude cell lysates
No full text3141Adeno-associated virus (AAV) based vectors have recently been gaining importance as DNA delivery systems. Efficient downstream processing of AAV remains a major challenge as serotypes differ in physicochemical properties, making it difficult to design uniform purification processes. Clarification of AAV is an especially critical step. Harvesting of AAV, like other viruses, often requires cell lysis, resulting in a difficult-to-filter cell lysate. In this study, we evaluated the applicability of diatomaceous earth (DE) as a filter aid for clarification of AAV crude cell lysates. DE filtration proved to be a viable clarification method for AAV2, AAV5 and AAV8. Based on a design of experiment approach, the DE concentration was identified as the main factor influencing AAV particle loss. The loss of AAV during DE filtration was limited to < 2% by maintaining the DE quantity below 0.181 mg DE/1010 AAV. Use of DE reduced manual handling time 3–fold and increased the filter capacity 3.5–fold compared to filtration combined with a prior centrifugation step. Moreover, we showed that the DE type had only a minor influence on the filtration performance. This study demonstrated that filtration with DE as a filter aid is an efficient clarification method for different AAV serotypes.36
A Novel and Simplified Anion Exchange Flow‐Through Polishing Approach for the Separation of Full From Empty Adeno‐Associated Virus Capsids
No full textABSTRACT Adeno‐associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome‐containing from empty AAV capsids, is usually time‐consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow‐through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl 2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4‐ to 7.5‐fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids’ functional potency and structural integrity. Following the successful establishment of the flow‐through polishing approach, it was adapted to a manual syringe‐based system. Manual flow‐through polishing using the monolith or membrane adsorber achieved 3.6‐ and 5.4‐fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow‐through polishing provides a rapid and easy‐to‐perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Combining steric exclusion with anion exchange - development of a universal and scalable adeno-associated virus downstream process
No full textAdeno-associated viruses (AAV) are among the leading vectors for in vivo gene therapy. The purification of AAV remains a bottleneck as it typically requires multiple individual process steps, often resulting in product loss and high costs. Current downstream processes are usually serotype-specific and rely primarily on expensive affinity resins. To address these limitations, we developed a serotype-independent purification method using steric exclusion chromatography (SXC) that can be combined with a subsequent anion exchange full/empty separation step. This alternative approach eliminates the need for intermediate concentration and buffer exchange, thereby reducing the number of process steps required while achieving high-purity full AAV particles. SXC conditions were optimized using a design of experiments approach. Isocratic separation of full and empty AAV resulted in further purification of the sample. The overall process achieved a viral genome recovery of 51.7 %, along with impurity depletions of 99.9 % for DNA and 99.8 % for protein. The process was successfully adapted to different AAV serotypes and genes of interest, demonstrating its robustness and versatility. In addition, the scalability of SXC was demonstrated, highlighting its potential for large-scale manufacturing. This streamlined, universal, and scalable process provides a robust and efficient alternative to traditional AAV purification processes, addressing critical challenges in gene therapy production and paving the way for broader implementation in research and manufacturing
Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes
No full textVariations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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