Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII.

INTRODUCTION: Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galß1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans. MATERIALS AND METHODS: We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection. RESULTS AND CONCLUSIONS: Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.

The first recombinant human coagulation factor VIII of human origin: human cell line and manufacturing characteristics.

INTRODUCTION: Since the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available. AIM: We describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product. METHODS AND RESULTS: A human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic. CONCLUSIONS: HEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety.

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium.

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.

A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion.

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.

Complete deletion of the neurotrophin receptor p75NTR leads to long-lasting increases in the number of basal forebrain cholinergic neurons.

Cholinergic neurons innervating cortical structures are among the most affected neuronal populations in Alzheimer's disease. In rodents, they express high levels of the neurotrophin receptor p75NTR. We have analyzed cholinergic septohippocampal neurons of the medial septal nucleus in p75exonIII (partial p75NTR knock-out) and p75exonIV (complete p75NTR knock-out) mice, in their original genetic background and in congenic strains. At postnatal day 15, the p75exonIII mutation leads to a moderate increase (+13%) in these neurons among littermates only after back-crossing in a C57BL/6 background. In contrast, the null p75exonIV mutation, which prevents expression of both the full-length and the shorter p75NTR isoforms, results in a 28% neuronal increase, independent of genetic background. The incomplete nature of the p75NTR mutation used previously, coupled with difficulties in delineating the mouse medial septum and the impact of the genetic background on cell numbers, all contribute to explain previous difficulties in establishing the role of p75NTR in regulating cholinergic neuron numbers in the mouse forebrain.