Improving transient gene expression in CHO-EBNA1 cells

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly generating recombinant proteins that are highly similar to those produced in stable CHO clones used for biomanufacturing. The higher cost of reagents necessary for TGE, specifically the requirement for large amounts of purified DNA and transfection agent for each production, means that improving the performance of CHO TGE could substantially augment the method’s utility. In the current study, we have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1). Transfection of EBNA-1-expressing cells with plasmid vectors encoding the Epstein-Barr virus OriP sequence boosted TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture in shake flasks, we optimized transfection parameters (plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added at 24 h post-transfection, followed by periodic glucose supplementation. This method gave a maximum yield of 900 mg/L (for the chimeric IgG4 B72-3 mAb), with an average of 570 mg/L (standard deviation: 250 mg/L) for a panel of six mAbs and 320 mg/L (standard deviation: 140 mg/L) for five His-tagged recombinant proteins at 14 days post-transfection. Compared to our current low-density TGE process using CHO-3E7 cells and different culture medium, the new procedure gave on average 3-fold higher yields; purified mAbs produced using the two methods had distinct glycosylation profiles (by HILIC analysis) but showed identical target binding kinetics by SPR. Key advantages of the improved CHO-3E7-based protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change

At the interface of community and healthcare systems: a longitudinal cohort study on evolving health and the impact of primary healthcare from the patient's perspectiv

<p>Abstract</p> <p>Background</p> <p>Massive efforts in Canada have been made to renew primary healthcare. However, although early evaluations of initiatives and research on certain aspects of the reform are promising, none have examined the link between patient assessments of care and health outcomes or the impacts at a population level. The goal of this project is to examine the effect of patient-centred and effective primary healthcare on the evolution of chronic illness burden and health functioning in a population, and in particularly vulnerable groups: the multi-morbid and the poor.</p> <p>Methods/Design</p> <p>A randomly selected cohort of 2000 adults aged 25 to 75 years will be recruited within the geographic boundaries of four local healthcare networks in Quebec. At recruitment, cohort members will report on socio-demographic information, functional health and healthcare use. Two weeks, 12 months and 24 months after recruitment, cohort participants will complete a self-administered questionnaire on current health and health behaviours in order to evaluate primary healthcare received in the previous year.</p> <p>The dependent variables are calculated as change over time of functional health status, chronic illness burden, and health behaviours. Dimensions of patient-centred care and clinical processes are measured using sub-scales of validated instruments. We will use Poisson regression modelling to estimate the incidence rate of chronic illness burden scores and structural equation modelling to explore relationships between variables and to examine the impact of dimensions of patient-centred care and effective primary healthcare.</p> <p>Discussion</p> <p>Results will provide valuable information for primary healthcare clinicians on the course of chronic illness over time and the impact on health outcomes of accessible, patient-centred and effective care. A demonstration of impact will contribute to the promotion of continuous quality improvement activities at a clinical level. While considerable advances have been made in the management of specific chronic illnesses, this will make a unique contribution to effective care for persons with multiple morbidities. Furthermore, the cohort and data architecture will serve as a research platform for future projects.</p

Suspension Vero cell culture technology for high titer production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71, hantaan, more recent COVID19 and other vaccines. Vero cell culture technologies were also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will significantly simplify scale-up and manufacturing processes. Development of advanced suspension Vero culture technology to improve product titer will further reduce the cost of goods. We previously reported a successful adaptation of adherent Vero cells originated from ATCC CCL-81 to grow in suspension in serum-free and animal component-free media developed in-house. The suspension adapted cells were found to retain their genetic stability and to be non-tumorigenic. Present work continues the development and optimization of cell culture process and feeding strategy to improve the growth of suspension Vero cell and the production of vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1). Data from this study showed the suspension adapted Vero cells retained similar VSV productivity to that obtained in adherent culture; volumetric productivity of VSV increased with the increasing cell density at infection in batch culture. However, the maximum cell density in batch culture was about 2.5x106 cells/mL, and was not improved significantly despite tremendous effort dedicated to improve culture conditions such as supplementing various nutrients in batch culture. As a result, perfusion culture was employed as an approach to increase cell density in the culture, which in turn increased the VSV productivity up to one log, at 1.1x1010 TCID50/mL when the culture infected at 7x106 cells/mL. High titer production of HSV-1 in the Vero culture is more challenging. The virus productivity is not only limited by the maximum cell density in batch culture, but also reduced by inhibitory metabolites secreted in the culture even at low cell density such as 1x106 cells/mL. Media replacement before virus infection is essential to achieve a high HSV-1 yield. As such, perfusion culture was a preferred mode for high titer production of HSV-1, which improved the HSV-1 titer also by up to one log to 1.8 x109 TCID50/ in a culture infected at 5x106 cells/mL when comparing to a control shake flask culture infected at 1x106 cells/mL. Experimental data also demonstrated that perfusion Vero culture was robust and reproducible. This study demonstrates that batch or perfusion suspension Vero culture is a much simplified process than current adherent culture technology for manufacturing of viral vaccines, and offers great potentials in reducing the cost of goods. The suspension Vero culture developed in our institute has generated tremendous interests from industry and academia, and are being tested by many different organizations

The QKI-6 and QKI-7 RNA Binding Proteins Block Proliferation and Promote Schwann Cell Myelination

BACKGROUND:The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS:To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE:Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3–induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB–FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness

What makes primary care effective for people in poverty living with multiple chronic conditions?: study protocol

Abstract Background: The inverse care law persists: people living in poverty have the greatest needs and face considerable challenges in getting the care they need. Evidence reveals that GPs encounter difficulties in delivering care to poor patients, while many of those patients feel stigmatized by healthcare professionals. Patients living in poverty report negative healthcare experiences and unmet healthcare needs. Indeed, there is a growing recognition in primary care research of the importance of addressing the capabilities and social conditions of the poor when delivering care. Few studies have looked at the factors contributing to effective and &quot;socially responsive&quot; care for people living in poverty. Methods/Design: Our study adopts a qualitative ethnographic approach in four healthcare organizations in deprived areas of metropolitan Montreal (Québec, Canada), using patient shadowing techniques and interviews. Data will be collected through fieldwork observations and informal interviews with patients before and after consultations. We will observe medical consultations, care organization activities, and waiting areas and reception of patients. We will conduct a total of 36 individual interviews with 12 GPs and 24 patients. The interviews will be audio-recorded and transcribed for purposes of analysis. The analysis consists of debriefing sessions, coding and interpretive analysis. Discussion: This study aims to investigate how positive healthcare interactions between physicians and patients can improve the management of chronic conditions. We hypothesize that factors related to care organization, to healthcare professionals&apos; experience and to patients may enhance the quality of healthcare interactions, which may have positive impacts for preventing and managing chronic conditions. Our study will provide a unique set of data grounded in the perspectives of healthcare professionals and of patients living in poverty

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

BACKGROUND: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. RESULTS: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. CONCLUSION: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b