Implementation of a process-scale adenovirus purification with a single-use platform

Adenovirus vectors are finding increasing application within the vaccine and gene therapy industries. Companies developing adenovirus-based biopharmaceuticals will benefit from single-use process platforms that are quick and easy to install and have been demonstrated previously to produce purified adenovirus. The platform can be adapted to new adenovirus products with a minimum investment of time and resource in process development activities. We have assembled a platform from existing and proven technologies, available from lab to production scale, that meet the quality requirements of the vaccine industry. The platforms includes clarification, ultrafiltration/diafiltration, chromatography and sterile filtration steps. The approach has been successfully demonstrated for the purification of an Adenovirus serotype 5 vector, at the 20 L scale. It allows the complete purification of the vaccine in one day

Membrane chromatography cassettes for bind and elute applications of viruses and large proteins

For flow-through polishing applications, membrane adsorbers have become a well-established technology. However, there is an increasing demand for bind and elute purifications for larger targets as adeno- and lentiviruses, virus like particles (VLP) and influenza. The reason is the higher binding capacity of macroporous membranes compared to conventional resins having much smaller pores and excluding them by size. But capture applications with such devices suffered from the current size limitation of 5 liters. Here we describe a modular cassette system which has been tested for scale-up and flow performance in comparison with void volume optimized capsules. The goals were to create a system up to 20 L membrane volume which can be optionally expanded to ~100 liter and, be able adapt exactly to the size needed (modular), using the same 4 and 8 mm bed height as the capsules and membranes for single- or intra batch re-use

Covering Chemical Diversity of Genetically-Modified Tomatoes Using Metabolomics for Objective Substantial Equivalence Assessment

As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms

A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins[W][OA]

This work identifies a region of the gibberellin receptor GID1 as being involved in gibberellin-dependent GID1–DELLA protein interaction and finds that the unique GA-independent DELLA protein interaction of a GID1 present in Arabidopsis and some other dicots also depends on this loop structure

Development of a Single-Step Antibody–Drug Conjugate Purification Process with Membrane Chromatography

Membrane chromatography is routinely used to remove host cell proteins, viral particles, and aggregates during antibody downstream processing. The application of membrane chromatography to the field of antibody-drug conjugates (ADCs) has been applied in a limited capacity and in only specialized scenarios. Here, we utilized the characteristics of the membrane adsorbers, Sartobind® S and Phenyl, for aggregate and payload clearance while polishing the ADC in a single chromatographic run. The Sartobind® S membrane was used in the removal of excess payload, while the Sartobind® Phenyl was used to polish the ADC by clearance of unwanted drug-to-antibody ratio (DAR) species and aggregates. The Sartobind® S membrane reproducibly achieved log-fold clearance of free payload with a 10 membrane-volume wash. Application of the Sartobind® Phenyl decreased aggregates and higher DAR species while increasing DAR homogeneity. The Sartobind® S and Phenyl membranes were placed in tandem to simplify the process in a single chromatographic run. With the optimized binding, washing, and elution conditions, the tandem membrane approach was performed in a shorter timescale with minimum solvent consumption and high yield. The application of the tandem membrane chromatography system presents a novel and efficient purification scheme that can be realized during ADC manufacturing

Protein Farnesylation on Nasopharyngeal Carcinoma, Molecular Background and Its Potential as a Therapeutic Target

Nasopharyngeal carcinoma (NPC) is one of the Epstein–Barr virus (EBV)-associated malignancies. NPC is highly metastatic compared to other head and neck carcinomas, and evidence has shown that the metastatic features of NPC are involved in EBV infection. The prognosis of advanced cases, especially those with distant metastasis, is still poor despite advancements in molecular research and its application to clinical settings. Thus, further advancement in basic and clinical research that may lead to novel therapeutic modalities is needed. Farnesylation is a lipid modification in the C-terminus of proteins. It enables proteins to attach to the lipid bilayer structure of cellular membranes. Farnesylation was initially identified as a key process of membrane association and activation of the RAS oncoprotein. Farnesylation is thus expected to be an ideal therapeutic target in anti-RAS therapy. Additionally, more and more molecular evidence has been reported, showing that proteins other than RAS are also farnesylated and have significant roles in cancer progression. However, although several clinical trials have been conducted in cancers with high rates of ras gene mutation, such as pancreatic carcinomas, the results were less favorable than anticipated. In contrast, favorable outcomes were reported in the results of a phase II trial on head and neck carcinoma. In this review, we provide an overview of the molecular pathogenesis of NPC in terms of the process of farnesylation and discuss the potential of anti-farnesylation therapy in the treatment of NPC

頸椎固定後の喉頭展開困難に対しMcGRATH MACを使用し二腔チューブの挿管が可能となった1症例

雑誌掲載版頸椎固定術後の喉頭展開困難症例に対しMcGRATH MACを使用し二腔チューブ(DLT)の挿管が可能となった1症例を経験した。前縦隔腫瘍の診断で胸腔鏡下腫瘍摘出術を予定していたが、頸部の可動域制限のため、マッキントッシュ喉頭鏡による喉頭展開時の視野はCormack-Lehane分類grade IIIであった。McGRATH MACの使用により視野はgrade Iと大幅に改善し、DLTの挿管を安全かつ容易に行うことができた。McGRATH MACは挿管困難症例のDLTの挿管の有用な選択肢であると考えられる。(著者抄録

Epstein–Barr Virus LMP1 Induces Soluble PD-L1 in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy. The principal oncogene of EBV, latent membrane protein 1 (LMP1), induces the expression of programmed death-ligand 1 (PD-L1), which is an immunosuppressive transmembrane protein and a promising therapeutic target for various malignancies. Recent studies have revealed an association between the level of soluble PD-L1 (sPD-L1) and disease progression. However, the role of sPD-L1 in NPC or its relevance to LMP1 has not been elucidated. This study aimed to examine whether LMP1 induces sPD-L1 in vitro and analyze the clinical relevance of LMP1, PD-L1, and sPD-L1 in NPC patients. Analysis of nasopharyngeal cell lines revealed that LMP1 induces both cellular PD-L1 and sPD-L1. Analysis of biopsy specimens from 32 NPC patients revealed that LMP1 expression was significantly correlated with PD-L1 expression. Finally, the serum sPD-L1 level in NPC patients was higher than that in the controls. Moreover, the sPD-L1 level in the advanced stage was higher than that in the early stage. However, LMP1 expression, PD-L1 expression, and sPD-L1 levels were not associated with prognosis. These results suggest that LMP1 induces both sPD-L1 and PD-L1, which are associated with NPC progression