A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

Artemin induced functional recovery and reinnervation after partial nerve injury.

Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.

Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches.

In developing biosimilar or biobetter products, comparability to the reference product is required to claim similar integrity or intended purpose. In this work, an anti-CD20 monoclonal antibody developed using RNA interference to decrease core fucosylation (RNAi-mediated) was comprehensively characterized by LC-MS and compared with the commercially-available anti-CD20 rituximab (MabThera (®) ). As anticipated, < 30% core fucose was found within the RNAi-produced molecule (compared with > 90% in rituximab), and the reduction in fucose resulting in a significant improvement in FcγRΙΙΙa binding and antibody-dependent cell-mediated cytotoxicity. Two mutations, S258Y (fully mutated) and F174I/L (partially mutated), however, were detected in the production of the RNAi-mediated molecule. An alternative LC-MS approach using dimethyl labeling (i.e., 2CH 2 for rituximab and 2CD 2 for the RNAi-mediated molecule) was developed to additionally compare the two mAbs and confirm the full sequence with the two mutation sites. Furthermore, disulfide linkages were found to be the same for the two antibodies, with a small portion of unpaired cysteines in both products. Disulfides were correctly linked if the samples were prepared at low pH (i.e., enzymatic digestion by pepsin at pH 2); however, trace amounts of scrambling were found by trypsin digestion at pH 6.8, and this scrambling increased significantly at pH 8. Typical modifications, such as pyro-Glu formation at the N-terminus, K clipping at the C-terminus, oxidation at Met, and deamidation at Asn, were also detected with no significant differences between the two products. Using the LC-MS approaches for the comparability study, product integrity with critical structure information was revealed for confirmation of intended purpose (core fucosylation), identification of critical parameters (e.g., sample pH), and correction as needed (amino acid mutation).

Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals.

Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.

Topographically specific regeneration of sensory axons in the spinal cord.

Artemin, a member of the glial-derived neurotrophic factor family, promotes robust regeneration of sensory axons after dorsal root crush. We report here that several classes of sensory axons regenerate to topographically appropriate regions of the dorsal horn with artemin treatment. Projections of regenerated muscle and cutaneous myelinated sensory afferents are restricted to the correct spinal segments and to appropriate regions within spinal gray matter. Regenerated unmyelinated axons expressing calcitonin gene-related peptide project only to superficial laminae of the dorsal horn, where uninjured nociceptive afferents project normally. In contrast, intraventricular infusion of a soluble form of the Nogo receptor that blocks the action of several myelin-associated inhibitory proteins promotes relatively unrestricted regeneration of sensory axons throughout the dorsal white and gray matter of the spinal cord. These results demonstrate that cues capable of guiding regenerating axons to appropriate spinal targets persist in the adult mammalian cord, but only some methods of stimulating regeneration allow the use of these cues by growing axons.

Stereochemistry-activity relationship of orally active tetralin S1P agonist prodrugs.

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

IGF-1:tetanus toxin fragment C fusion protein improves delivery of IGF-1 to spinal cord but fails to prolong survival of ALS mice.

To improve delivery of human insulin-like growth factor-1 (hIGF-1) to brain and spinal cord, we generated a soluble IGF-1:tetanus toxin fragment C fusion protein (IGF-1:TTC) as a secreted product from insect cells. IGF-1:TTC exhibited IGF-1 and TTC activity in vitro; it increased levels of immunoreactive phosphoAkt in treated MCF-7 cells and bound to immobilized ganglioside GT1b. In mice, the fusion protein underwent retrograde transport by spinal cord motor neurons following intramuscular injection, and exhibited both TTC- and IGF-1 activity in the CNS following intrathecal infusion. Analogous to the case with TTC, intrathecal infusion of the fusion protein resulted in substantial levels of IGF-1:TTC in spinal cord tissue extracts. Tissue concentrations of hIGF-1 in lumbar spinal cords of mice infused with IGF-1:TTC were estimated to be approximately 500-fold higher than those in mice treated with unmodified recombinant hIGF-1 (rhIGF-1). Like rhIGF-1, infusion of IGF-1:TTC reduced levels of IGF-1 receptor immunoreactivity in the same extracts. Despite raising levels of exogenous hIGF-1 in spinal cord, intramuscular- or intrathecal administration of IGF-1:TTC had no significant effect on disease progression or survival of high-expressing SOD1(G93A) transgenic mice. IGF-1:TTC may prove to be neuroprotective in other animal models of CNS disease or injury known to be responsive to unmodified IGF-1.

Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury.

Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.

Artemin crystal structure reveals insights into heparan sulfate binding.

Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFRalpha3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangement characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.

New approaches for the treatment of pain: the GDNF family of neurotrophic growth factors.

This article focuses on the GDNF family of neurotrophic factors as a potential new class of therapeutics for neuropathic pain, with a particular emphasis on the ligands, artemin and GDNF. In vivo activity of the ligands, expression of ligands and receptors after peripheral nerve injury, and modulation of nerve injury-induced changes by the ligands are reviewed in detail. Structural considerations, particularly with regard to implications for binding interactions and biological activity are discussed.

Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin.

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.

Multiple actions of systemic artemin in experimental neuropathy.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.