Louis Bisson

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

BackgroundMammalian 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.ResultsWe 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.ConclusionThese results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.

Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusia ni High‐Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.

Plasminogen Kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways

Proteolytic processing of human plasminogen generates potent antiangiogenic peptides such as angiostatin. The plasminogen kringle 5 (K5) domain, which is distinct from angiostatin, possesses potent antiangiogenic properties on its own, which can be exploited in cancer therapy. It has been recently observed that antiangiogenic agents promote leukocyte-vessel wall interaction as part of their antitumor effect. Although we have previously shown that K5 suppresses cancer growth in tumor xenograft models, its modulation of inflammation in experimental mice with intact immune systems is unknown. To determine whether K5 possesses immune proinflammatory properties, we investigated the effects of K5 in an immune competent model of breast cancer and observed that tumor rejection is substantially reduced in nonobese diabetic/severe combined immunodeficient and BALB/c nude when compared with wild-type BALB/c mice, suggesting an important role for T-lymphoid cells in the antitumor effect of K5. Tumor explant analysis shows that K5 enhances tumor recruitment of CD3+ lymphoid cells, in particular, the NKT phenotype. We also observed a significant decrease in tumor-associated microvessel length and density consistent with antiangiogenic activity. Histologic analysis of K5 tumors also revealed a robust neutrophilic infiltrate, which may be explained by the neutrophil chemotactic activity of K5 as well as its ability to promote CD64 up-regulation within the CD11b+ adhesive neutrophil population. In sum, our findings confirm that the K5 protein acts as a potent angiostatic agent and possesses a novel proinflammatory role via its ability to recruit tumor-associated neutrophils and NKT lymphocytes, leading to a potent antitumor response. [Mol Cancer Ther 2007;6(2):441–9]

Stable expression of chimeric heavy chain antibodies in CHO cells.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

Purification of His-Tagged Proteins Using Fractogel-Cobalt

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Such proteins often need to be as pure as possible before any characterization study can begin. Although many types of protein tag are available, histidine is the most popular. Although small-scale immobilized metal-affinity column (IMAC) purification of such proteins (e.g., <500 mL of culture medium) can easily be achieved using gravity chromatography columns, larger volumes can be processed with the aid of automated chromatography systems. This protocol describes an IMAC purification technique for secreted proteins using a cobalt-loaded resin. Preliminary small-scale trials using this technique can be used to determine the production scale that will be needed to provide enough pure material for a given study.