Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering.

Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.

Increased potency of recombinant VWF D'D3 albumin fusion proteins engineered for enhanced affinity for coagulation factor VIII.

BACKGROUND: We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES: The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS: Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS: In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS: In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

Reprint of: Expression and purification of ataxin-1 protein.

Ataxin-1 is part of a larger family of polyglutamine-containing proteins that is linked to nine distinct neurodegenerative disorders. There are no known effective therapies for any of these expanded polyglutamine tract disorders. One possible reason for this is the lack of sufficient amounts of pure polyglutamine-containing proteins suitable for biochemical and conformational studies. Here, we show that we were able to successfully purify a non-pathological, wild-type human ataxin-1 protein containing a 30-glutamine repeat sequence. This ataxin-1 protein was expressed in Escherichia coli as a fusion protein with a GST tag at the N-terminus and a double (His)(6) tag at the C-terminus. The devised dual affinity tag strategy allowed successful purification of the full-length ataxin-1 fusion protein to 90% homogeneity as confirmed by Western blot analysis using the two monoclonal ataxin-1 antibodies developed in our laboratory. In addition, the GST tag was successfully removed from the purified ataxin-1 fusion protein by treatment with Tobacco etch virus (TEV) protease. Since polyglutamine-containing proteins tend to aggregate, solvents/buffers that minimize aggregation have been used in the purification process. This dual affinity purification protocol could serve as a useful basis for purifying aggregation-prone proteins that are involved in other neurodegenerative diseases.

Expression and purification of ataxin-1 protein.

Ataxin-1 is part of a larger family of polyglutamine-containing proteins that is linked to nine distinct neurodegenerative disorders. There are no known effective therapies for any of these expanded polyglutamine tract disorders. One possible reason for this is the lack of sufficient amounts of pure polyglutamine-containing proteins suitable for biochemical and conformational studies. Here, we show that we were able to successfully purify a non-pathological, wild-type human ataxin-1 protein containing a 30-glutamine repeat sequence. This ataxin-1 protein was expressed in Escherichia coli as a fusion protein with a GST tag at the N-terminus and a double (His)(6) tag at the C-terminus. The devised dual affinity tag strategy allowed successful purification of the full-length ataxin-1 fusion protein to 90% homogeneity as confirmed by Western blot analysis using the two monoclonal ataxin-1 antibodies developed in our laboratory. In addition, the GST tag was successfully removed from the purified ataxin-1 fusion protein by treatment with Tobacco etch virus (TEV) protease. Since polyglutamine-containing proteins tend to aggregate, solvents/buffers that minimize aggregation have been used in the purification process. This dual affinity purification protocol could serve as a useful basis for purifying aggregation-prone proteins that are involved in other neurodegenerative diseases.

Inclusion formation by ataxins -1, -2, -3, and -7.

The authors studied inclusion formation in vitro using transiently transfected PC12 cells, with epitope-tagged and untagged full-length and truncated wild-type and expanded ataxins -1, -2, -3, and -7. At 72 hours, no inclusions were seen with wild-type full-length or truncated ataxins -2, -3, or -7, and only one with ataxin-1. Truncation abolished nuclear localization of ataxins -1 and -7, and allowed nuclear entry of ataxin-2. Of the expanded ataxins, only -1 and -2 formed inclusions, and those of ataxin-2 were rare and exclusively cytoplasmic. Truncation resulted in inclusion formation by ataxins -3 and -7, increased ataxin-1 inclusions, and enabled formation of nuclear ataxin-2 inclusions. There was no recruitment of wild-type ataxin-1 to expanded ataxin-1 inclusions.

Different ataxin-2 antibodies display different immunoreactive profiles.

We have developed a monoclonal antibody (4A7) directed against the C-terminus of the ataxin-2 protein that is involved in the polyglutamine neurodegenerative disorder spinocerebellar ataxia type 2. Comparison with other ataxin-2 antibodies showed that 4A7 specifically recognized ataxin-2. In contrast, a previously reported ataxin-2 antibody (15F6) did not appear to recognize full-length ataxin-2 in our systems. Immunocytochemical and subcellular fractionation studies using 4A7 confirmed previous reports that ataxin-2 is localized to both the cytoplasm and the trans-Golgi network in rat PC12 cells and rat brain tissue. In contrast, 4A7 failed to label the trans-Golgi network in the three primate cell lines examined. Cytoplasmic ataxin-2 was not associated with mitochondria, lysosomes, endoplasmic reticulum, peroxisomes, proteasomes, clathrin-coated pits or vesicles, or F-actin. Ataxin-2 was found to be phosphorylated but not glycosylated, and exhibited an estimated half-life of not less than 21 h. Interestingly, another commercially available ataxin-2 antibody did not detect ataxin-2 localized to the trans-Golgi network. This antibody was also found to immunoprecipitate fewer proteins/protein partners than 4A7. Although cross-reactivity of the 4A7 antibody with other protein(s) cannot be ruled out, it appears likely that the interaction of ataxin-2 with other cell components is dependent on both the host cell type and its subsequent subcellular localization.