Process intensification through integration of upstream perfusion cell culture with downstream continuous chromatography in monoclonal antibody production

Process intensification is gaining interest as a strategy to reduce production costs, while improving product quality and throughput in the manufacturing of biopharmaceuticals. For a competitive production process, continuous or semi-continuous upstream and downstream processing can be employed. Compared with a process performed in batch runs, continuous processing allows for increased capacity utilization and eliminates or minimized the need for intermediate hold-up steps. Here, we describe the integration of a high-performing upstream cell culture process with downstream purification utilizing new emerging technologies such as periodic counter-current (PCC) chromatography and straight-through processing (STP). A high-cell density perfusion process based on commercially available ActiCHO™ cell culture media was developed for a MAb-producing Chinese hamster ovary (CHO) cell line. Medium prototypes were evaluated in small scale using the single-use ReadyToProcess WAVE™ 25 bioreactor system. Medium optimization resulted in a final process with a cell-specific perfusion rate (CSPR) of less than 50 pL/cell/d. Process performance was verified at laboratory scale using single-use stirred-tank bioreactor systems. Productivity and product quality of the developed perfusion process were compared with a standard fed-batch process. MAb capture on MabSelect SuRe™ LX protein A chromatography medium (resin) was performed in a three-column PCC (3C PCC) setup. The capture step was followed by two polishing steps using Capto™ S ImpAct ion exchange and Capto adhere multimodal ion exchange media in serially connected columns in a STP setup. MAb purity and yield of the developed continuous processes were compared with traditional setups performed in batch runs. In this case study, we demonstrate the feasibility of integrated upstream and downstream MAb processing performed in a continuous manner. The developed process shows a performance equivalent to traditional processing performed in batch runs

Efficient approaches for perfusion medium development

Here, we present a fast and convenient strategy for developing a high-cell density perfusion process for antibody-producing Chinese hamster ovary (CHO) cells based on the commercially available ActiCHO™ Media System. ActiCHO P base medium was used as a starting point and ActiCHO Feed-A and Feed-B were added in various concentrations as supplements. The resulting perfusion medium prototypes were first evaluated in batch cultures, applying a design of experiment (DoE) strategy (Figure 1), and then tested in small-scale perfusion cultures in rocking single-use WAVE bioreactor™ systems (Figure 2). The medium optimization resulted in a final process with a cell-specific perfusion rate (CSPR) of less than 50 pL/cell/d, which is a more than 45% decrease compared with the starting process conditions. The performance of the perfusion process was further validated in lab-scale single-use stirred-tank bioreactor systems. Productivity and product quality of the perfusion process were compared with a standard fed-batch culture process

Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy

The perihydroxylated perylene quinone hypericin has been reported to possess potent anti-metastatic and antiangiogenic activities, generated by targeting diverse crossroads of cancer-promoting processes via unique mechanisms. Hypericin is the only known exogenous reagent that can induce forced poly-ubiquitination and accelerated degradation of heat shock protein 90 (Hsp90) in cancer cells. Hsp90 client proteins are thereby destabilized and rapidly degraded. Hsp70 client proteins may potentially be also affected via preventing formation of hsp90-hsp70 intermediate complexes. We show here that hypericin also induces enhanced degradation of hypoxia-inducible factor 1α (HIF-1α) in two human tumor cell lines, U87-MG glioblastoma and RCC-C2VHL−/− renal cell carcinoma and in the non-malignant ARPE19 retinal pigment epithelial cell line. The hypericin-accelerated turnover of HIF-1α, the regulatory precursor of the HIF-1 transcription factor which promotes hypoxic stress and angiogenic responses, overcomes the physiologic HIF-1α protein stabilization which occurs in hypoxic cells. The hypericin effect also eliminates the high HIF-1α levels expressed constitutively in the von-Hippel Lindau protein (pVHL)-deficient RCC-C2VHL−/− renal cell carcinoma cell line. Unlike the normal ubiquitin-proteasome pathway-dependent turnover of HIF-α proteins which occurs in normoxia, the hypericin-induced HIF-1α catabolism can occur independently of cellular oxygen levels or pVHL-promoted ubiquitin ligation of HIF-1α. It is mediated by lysosomal cathepsin-B enzymes with cathepsin-B activity being optimized in the cells through hypericin-mediated reduction in intracellular pH. Our findings suggest that hypericin may potentially be useful in preventing growth of tumors in which HIF-1α plays pivotal roles, and in pVHL ablated tumor cells such as renal cell carcinoma through elimination of elevated HIF-1α contents in these cells, scaling down the excessive angiogenesis which characterizes these tumors

Scheduling and routing of agvs for large-scale flexible manufacturing systems

In this paper, we propose a new heuristic as well as several improvements to an existing approach based on Benders decomposition for solving the conflict free scheduling and routing of automated guided vehicles (AGVs), with promising results. The existing method solves the problem in two stages; task assignment/sequencing, and feasibility check of the first stage\u27s solution subject to collision-avoidance constraints. The method is not suitable for large-scale AGV systems. We proposed several improvements and speedup strategies that result in fast methods capable of scheduling AGVs in a realistic layout with a graph of several hundred nodes and arcs. This is done by reformulating the mathematical model of the problem. We also introduce a new heuristic based on the improved method that yields high-quality solutions quickly. Moreover, we solve a real large-scale industrial instance by a commercial constraint programming solver and an open-source SMT solver. The results show that both of these general-purpose solvers can effectively solve our proposed models

General practice training and virtual communities of practice - a review of the literature

<p>Abstract</p> <p>Background</p> <p>Good General Practice is essential for an effective health system. Good General Practice training is essential to sustain the workforce, however training for General Practice can be hampered by a number of pressures, including professional, structural and social isolation. General Practice trainees may be under more pressure than fully registered General Practitioners, and yet isolation can lead doctors to reduce hours and move away from rural practice. Virtual communities of practice (VCoPs) in business have been shown to be effective in improving knowledge sharing, thus reducing professional and structural isolation. This literature review will critically examine the current evidence relevant to virtual communities of practice in General Practice training, identify evidence-based principles that might guide their construction and suggest further avenues for research.</p> <p>Methods</p> <p>Major online databases <it>Scopus</it>, <it>Psychlit</it> and <it>Pubmed</it> were searched for the terms “Community of Practice” (CoP) AND (Online OR Virtual OR Electronic) AND (health OR healthcare OR medicine OR “Allied Health”). Only peer-reviewed journal articles in English were selected. A total of 76 articles were identified, with 23 meeting the inclusion criteria. There were no studies on CoP or VCoP in General Practice training. The review was structured using a framework of six themes for establishing communities of practice, derived from a key study from the business literature. This framework has been used to analyse the literature to determine whether similar themes are present in the health literature and to identify evidence in support of virtual communities of practice for General Practice training.</p> <p>Results</p> <p>The framework developed by Probst is mirrored in the health literature, albeit with some variations. In particular the roles of facilitator or moderator and leader whilst overlapping, are different. VCoPs are usually collaborations between stakeholders rather than single company VCoPs. Specific goals are important, but in specialised health fields sometimes less important than in business. Boundary spanning can involve the interactions of different professional groups, as well as using external experts seen in business VCoPs. There was less use of measurement in health VCoPs. Environments must be supportive as well as risk free. Additional findings were that ease of use of technology is paramount and it is desirable for VCoPs to blend online and face-to-face involvement.</p> <p>Conclusions</p> <p>The business themes of leadership, sponsorship, objectives and goals, boundary spanning, risk-free environment and measurements become, in the health literature, facilitation, champion and support, objectives and goals, a broad church, supportive environment, measurement benchmarking and feedback, and technology and community.</p> <p>General Practice training is under pressure from isolation and virtual communities of practice may be a way of overcoming isolation. The health literature supports, with some variation, the business CoP framework developed by Probst. Further research is needed to clarify whether this framework is an effective method of health VCoP development and if these VCoPs overcome isolation and thus improve rural retention of General Practice registrars.</p