Evaluation of novel CEX resin for continous processing of MAb purification

We have evaluated novel cation exchange resin and membrane which is potentially suited for continuous process in biologics manufacturing. Typically cation exchange chromatography (CEX) is used in a bind/elute (B/E) mode for the MAb process since MAb act as cation at the buffer condition used for CEX due to the pI from neutral to weak basic. Generally, purpose of CEX chromatography is to remove aggregates and other impurities like HCP and DNA. Especially, aggregate removal is of interest to the industries. A continuous process in MAb downstream process can solve several bottlenecks in typical batch process. However its capability cannot be fully utilized if some step of chromatography is used in a B/E mode since it will take a longer process time due to the posing of product stream for binding and washing and it will take a more cost due to the requirement of large amount of resins as we use at the batch process. In such a case, chromatography in flowthrough mode has a potential to overcome those issues for both cost and time and enables us to develop more streamlined continuous process. We will present the result of our study to evaluate novel CEX resin developed by Merck Millipore by comparing with the existing process of Mab A and we obtained a conclusion that this resin is fitted to the continuous processing very much. The novel resin showed a better impurity clearance than our existing process. For example, over 65% removal of aggregate by the novel resin was obtained in contrast to no removal by the existing process. A 20-fold better clearance for DNA was confirmed for the novel resin than the existing process. This indicates an additional polishing step can be omitted and this new chromatography can be a strong option if we need to reduce such impurities further. Also the resin cost is expected to be reduced down to 1/10 in maximum since those impurity clearance results were obtained about 10-times larger load than the typical B/E mode operation of CEX. Considering those aspects, we conclude that this resin showed a better fit for a continuous process. We will also discuss an expected effect of the novel CEX resin on cost and process time savings by a continuous proces

Measurements of neutron total and capture cross sections of 139^{139}La and evaluation of resonance parameters

Neutron total and capture cross sections of Lanthanum(La)-139 were measured at the Accurate Ne-utron-Nucleus Reaction measurement Instrument (ANNRI) of the Materials and Life Science Experimental Facility (MLF) in the Japan Proton Accelerator Research Complex (J-PARC). The total cross section was largely different from that in evaluated libraries, such as JENDL-5, in the energy range from 80 to 900~eV. Resonance parameters for four resonances including one negative resonance were obtained using a resonance analysis code, REFIT. The resonance analysis revealed discrepancies in several resonance parameters with the evaluated libraries. Furthermore, the information about the scattering radius was also extracted from the results of the total cross section. The obtained scattering radius was larger than that recorded in the evaluated libraries.Comment: 12 pages, 16 figure

How to Make a Secure Index for Searchable Symmetric Encryption, Revisited

Searchable symmetric encryption (SSE) enables clients to search encrypted data. Curtmola et al. (ACM CCS 2006) formalized a model and security notions of SSE and proposed two concrete constructions called SSE-1 and SSE-2. After the seminal work by Curtmola et al., SSE becomes an active area of encrypted search. In this paper, we focus on two unnoticed problems in the seminal paper by Curtmola et al. First, we show that SSE-2 does not appropriately implement Curtmola et al.\u27s construction idea for dummy addition. We refine SSE-2\u27s (and its variants\u27) dummy-adding procedure to keep the number of dummies sufficiently many but as small as possible. We then show how to extend it to the dynamic setting while keeping the dummy-adding procedure work well and implement our scheme to show its practical efficiency. Second, we point out that the SSE-1 can cause a search error when a searched keyword is not contained in any document file stored at a server and show how to fix it

Donor Treg expansion by liposomal alpha-galactosylceramide modulates Tfh cells and prevents sclerodermatous chronic graft-versus-host disease

Background and Aim: Chronic graft-versus-host disease (cGVHD) is a major cause of nonrelapse morbidity and mortality following hematopoietic stem cell transplantation (HSCT). alpha-Galactosylceramide (alpha-GC) is a synthetic glycolipid that is recognized by the invariant T-cell receptor of invariant natural killer T (iNKT) cells in a CD1d-restricted manner. Stimulation of iNKT cells by alpha-GC leads to the production of not only immune-stimulatory cytokines but also immune-regulatory cytokines followed by regulatory T-cell (Treg) expansion in vivo. Methods: We investigated the effect of iNKT stimulation by liposomal alpha-GC just after transplant on the subsequent immune reconstitution and the development of sclerodermatous cGVHD. Results: Our study showed that multiple administrations of liposomal alpha-GC modulated both host- and donor-derived iNKT cell homeostasis and induced an early expansion of donor Tregs. We also demonstrated that the immune modulation of the acute phase was followed by the decreased levels of CXCL13 in plasma and follicular helper T cells in lymph nodes, which inhibited germinal center formation, resulting in the efficient prevention of sclerodermatous cGVHD. Conclusions: These data demonstrated an important coordination of T- and B-cell immunity in the pathogenesis of cGVHD and may provide a novel clinical strategy for the induction of immune tolerance after allogeneic HSCT

Reduced dose of PTCy followed by adjuvant alpha-galactosylceramide enhances GVL effect without sacrificing GVHD suppression

Posttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient's condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by alpha -galactosylceramide (alpha -GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of alpha -GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4(+)Foxp3(+) regulatory T cells. These studies indicate that alpha -GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk

Hematopoietic stem cell-derived Tregs are essential for maintaining favorable B cell lymphopoiesis following posttransplant cyclophosphamide

Posttransplant cyclophosphamide (PTCy) is associated with a low incidence of chronic graft -versus-host disease (cGVHD) following hematopoietic stem cell (HSC) transplantation. Previous studies have shown the important roles of B cell immunity in cGVHD development. Here, we investigated the long-term reconstitution of B lymphopoiesis after PTCy using murine models. We first demonstrated that the immune homeostatic abnormality leading to cGVHD is characterized by an initial increase in effector T cells in the bone marrow and subsequent B and Treg cytopenia. PTCy, but not cyclosporine A or rapamycin, inhibits the initial alloreactive T cell response, which restores intra-bone marrow B lymphogenesis with a concomitant vigorous increase in Tregs. This leads to profound changes in posttransplant B cell homeostasis, including decreased B cell activating factors, increased transitional and regulatory B cells, and decreased germinal center B cells. To identify the cells responsible for PTCy-induced B cell tolerance, we selectively depleted Treg populations that were graft or HSC derived using DEREG mice. Deletion of either Treg population without PTCy resulted in critical B cytopenia. PTCy rescued B lymphopoiesis from graft-derived Treg deletion. In contrast, the negative effect of HSC-derived Treg deletion could not be overcome by PTCy, indicating that HSC-derived Tregs are essential for maintaining favorable B lymphopoiesis following PTCy. These findings define the mechanisms by which PTCy restores homeostasis of the B cell lineage and reestablishes immune tolerance

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

IL-1b signaling augments continued splenic monocyte supply during acute inflammation