The merits of continuous processing over batch processing are well known in the manufacturing industry. Continuous operation results in shorter process times due to omission of hold steps, higher productivity due to reduced shutdown costs, and lowers labor requirement. Over the past decade, there has been an increasing interest in continuous processing within the bioprocessing community, specifically those involved in production of biotherapeutics. Continuous operations in upstream processing (perfusion) have been performed for decades. However, recent development of continuous downstream operations has led the industry to envisage an integrated bioprocessing platform for efficient production. The regulators, key players in the biotherapeutic industry, have also expressed their interest and willingness in this migration from the traditional batch processing. This paper aims to review major developments in continuous bioprocessing in the past decade. A discussion of pros and cons of the different proposed approaches has also been presented.

The intestinal epithelial lining is a very dynamic interface, where multiple interactions occur with the external world. The intestinal epithelial barrier is continuously exposed to a huge load of commensal microorganisms, food-borne antigens, as well as invading enteropathogens. Intestinal epithelial cells (IECs) and underlying immune cells are the main players in maintaining the delicate balance between gut tolerance and inflammation. IECs deferentially express the variety of chemokines and chemokine receptors, and these receptor-ligand interactions not only mediate the infiltration and activation of immune cells but also switch on the survival cascades in IECs. In this review, we discussed how chemokine-chemokine receptor-induced interactions play a central role to coordinate the interplay between IECs and gut immune cells to maintain homeostasis or elicit gut inflammation. Furthermore, we discussed how chemokines and chemokine receptors were used as a target for developing new drugs and therapies to control gut inflammation and autoimmunity.

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Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

The efficiency of the protein refolding process lies in identification of the optimal conditions. However, a number of challenges need to be overcome to achieve this. This review first describes the protein refolding process that is utilized presently for production of protein therapeutics. Next, it discusses the various shortcomings that exist with respect to the present approach. The focus of the paper is on presentation of the significant advancements that have been made in the past decade in the various aspects of protein folding, including use of bioinformatics, mechanistic modeling, analytical monitoring, process optimization, use of additives, high throughput development, on‐column refolding, Quality by Design (QbD), Process Analytical Technology (PAT), and process intensification. Finally, an approach is proposed that incorporates the best practices that have been identified in the various areas. The paper is expected to be of interest to those in academia and industry working in the area of protein refolding. © 2013 Society of Chemical Industry.

BackgroundThe trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.Methodology and Principal FindingsIn the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.Conclusions/SignificanceThe present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.

The chemokine receptor CCR9 and its only known ligand CCL25 play an important role in gut inflammation and autoimmune colitis. The function of CCR9‐CCL25 in the migration of immune cells is well characterized. However, its role in the immune cell differentiation is mostly not known. Using dextran sodium sulfate (DSS)‐induced gut inflammation model, we showed that CCR9+ dendritic cells (DCs) specifically CD11b−CD103+ DCs were significantly increased in the gut‐associated lymphoid tissues (GALT) compared to control mice. These CCR9+ DCs express lower MHC II and CD86 molecules and had regulatory surface markers (FasL and latency‐associated peptide, LAP) in the GALT. In the presence of CCL25, CCR9+ DCs promoted in vitro differentiation of Foxp3+ regulatory CD4+ T cells (Tregs). CCL25‐induced differentiation of Tregs was due to intrinsic signaling in the DCs but not through CD4+ T cells, which was driven by the production of thymic stromal lymphopoietin (TSLP) and not IL‐10. Furthermore, adoptive transfer of CCR9+ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the OVA‐specific gut‐allergic response. Our results suggest CCR9+ DCs have a regulatory function and may provide a new cellular therapeutic strategy to control gut inflammation and allergic immune reaction.

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