Comparison of platform host cell protein ELISA to process-specific host cell protein ELISA.

During expression of biotherapeutic proteins, complex mixtures of additional proteins are also produced by normal expression machinery of the host cell (termed "host cell proteins," or HCP). HCPs pose a potential impact to patient safety and product efficacy, and therefore must be well-characterized and the ability of the process to clear these proteins must be demonstrated. Due to the complexity of HCP, the method(s) used for monitoring must be demonstrated to provide sufficient information about relevant proteins.

Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D.

More similar than different: Host cell protein production using three null CHO cell lines.

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product.

Host cell protein testing by ELISAs and the use of orthogonal methods.

Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing.

2-o-phosphorylation of xylose and 6-o-sulfation of galactose in the protein linkage region of glycosaminoglycans influence the glucuronyltransferase-I activity involved in the linkage region synthesis.

Sulfated glycosaminoglycans (GAGs), including heparan sulfate and chondroitin sulfate, are synthesized on the so-called common GAG-protein linkage region (GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser) of core proteins, which is formed by the stepwise addition of monosaccharide residues by the respective specific glycosyltransferases. Glucuronyltransferase-I (GlcAT-I) is the key enzyme that completes the synthesis of this linkage region, which is a prerequisite for the conversion of core proteins to functional proteoglycans bearing GAGs. The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively, although the biological significance of these modifications remains to be clarified.

Proteomic profiling of recombinant Escherichia coli in high-cell-density fermentations for improved production of an antibody fragment biopharmaceutical.

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E.

High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain.

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases.

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations.

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells.

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain.

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng.

[Bilateral carotid stenting for bilateral carotid artery stenosis improved vascular dementia].

We report a case of bilateral internal carotid artery (ICA) stenosis treated with stenting. A 78-year-old man suffered from vascular dementia and left hemiparesis, and, by magnetic resonance angiogram (MRA), was diagnosed as having bilateral ICA stenosis. Cerebral angiogram showed severe, bilateral ICA stenosis (right; 88%, left; 93%) and xenon single photon emission tomography (SPECT) showed severely decreased cerebral blood flow (CBF) and cerebrovascular reactivity (CVR).

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes.

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein.

Synthesis of phosphorylated and sulfated glycosyl serines in the linkage region of the glycosaminoglycans.

We synthesized novel acidic glycans having acidic groups located in the linkage region of the glycosaminoglycans (GAGs). The targeted compounds, beta-D-Xyl(2P)-Ser (1), beta-D-Gal(+/-6S)-(1-->4)-beta-D-Xyl(2P)-Ser (3 and 2), beta-D-Gal(+/-6S)-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl(2P)-Ser (5 and 4), and beta-D-Gal-(1-->3)-beta-D-Gal(6S)-(1-->4)-beta-D-Xyl(2P)-Ser (6) contain phosphate and/or sulfate at the specified positions. Some of them (3, 5, and 6) are the first synthesized examples of natural-type glycoconjugates that simultaneously possess phosphate and sulfate as well as carboxylic acid.

Stereoselective syntheses of phosphorylated and sulfated glycosyl serines in glycosaminoglycan for biological probes.

Phosphorylated glycosyl serines of glycosaminoglycan with/without sulfate: beta-D-Xyl(2P)-Ser (1) and beta-D-Gal(+/-6S)-(1->4)-beta-D-Xyl(2P)-Ser (2, 3) were suitably designed for biological probes. These oligosaccharides were synthesized in a stereocontrolled manner.

Distribution and morphology of valves in the human external jugular vein: indications for utilization in microvascular anastomosis.

Purpose: This study examined the valves in the external jugular vein (EJV) that could affect microvascular anastomosis and increase the risk of thrombogenesis.

Materials And Methods: The EJVs of 57 sides from 36 cadavers aged 40 to 95 years (20 males and 16 females) were dissected. After opening the veins, the distribution and morphology of the valves were investigated using a stereoscopic microscope.

Anatomical study on valves of human facial veins.

The valves of the facial veins of 39 sides from 20 cadavers, aged 54-95 years, were investigated. Anatomical study of the valves is important, because the facial veins can be used for microvascular anastomosis in reconstruction in the head and neck. The present study confirmed that valves existed in the human facial veins, and were most frequently observed around the lower border of the mandible.

Possible mechanism to induce protein kinase C-dependent arterial smooth muscle contraction after subarachnoid haemorrhage.

A possible mechanism for the induction of protein kinase C (PKC)-dependent vascular contraction independent to the increase of intracellular Ca++ was investigated in the pathogenesis of cerebral vasospasm in the double subarachnoid haemorrhage (SAH) model. The level of 1,2-diacylglycerol (DAG), which is an intrinsic PKC activator, significantly increased from days 4 to 7 in the basilar artery after the initial SAH, and the continuous administration of 1,2-bis(nicotinamido)-propane (AVS), a novel free radical scavenger, not only lowered the concentration of lipid peroxides in the CSF but also successfully suppressed the basilar arterial wall in the same model. It was suggested that lipid peroxides generated in the subarachnoid clot affect the DAG content of the cerebral artery.

Involvement of oxygen radicals in the differentiation of rat myelomonocytic leukemia cells in vitro and in vivo.

We have found that LPS induces the differentiation of an LPS-resistant subline (LR) of rat myelomonocytic leukemia cell line, c-WRT-7, in vivo, which are resistant to the differentiation inducing effects of LPS in vitro. Furthermore, we have found that the differentiation of LR cells induced by LPS is inhibited by superoxide dismutase, which is one of radical scavengers. Accordingly, we have examined the differentiation inducing effects of xanthine oxidase, a potential source of oxygen radicals, on LR cells in vitro and in vivo.

Identification of ionizable groups essential for the enzyme catalysis on glutathione S-transferase P.

The pH-Vmax/KmGSH plot of glutathione S-transferase P (GST-P) showed a bell-shaped profile, indicating bifunctional catalysis for glutathione (GSH) conjugation. The ionization constant (Ke) and the heat of ionization (delta He) of the essential ionizable group in the GSH binding site were measured and the value of pKe1 was 5.9 and that of pKe2, 8.

A case of distal anterior choroidal artery aneurysm: case report and review of the literature.

We describe a very rare case of distal anterior choroidal artery aneurysm. A 34-year-old woman was admitted to our hospital because of headache and vomiting. Computed tomographic scans revealed a diffuse intraventricular hematoma and a thin subarachnoid hemorrhage.

Role of protein kinase C in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage.

This study investigated the role of protein kinase C (PKC) in the pathogenesis of vasospasm after experimental subarachnoid hemorrhage (SAH). PKC activation by intracisternal injection of a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TP)] induced dose-dependent, slowly developing, severe contraction of the basilar artery. A single intracisternal injection of TP (5 x 10(-9) M in the CSF) induced sustained contraction lasting over 3 days, which almost paralleled the changes of membrane-bound PKC activity in the basilar arterial wall.

Ecological structure and incidence rates of schizophrenia in Nagasaki City.

The purpose of this study was to investigate the relationship between incidence rates of schizophrenia and residential features in Nagasaki City, Japan. Social features of residents in 259 areas in the Nagasaki City ecological structure were determined using the results of the 1980 national census. Six residential factors--employment status, occupational and academic career, household status, length of residence, housing tenure and young male predominance--were selected as factors that clearly reflect residential differentiation of residents.

[A clinicopathological study of the spinal leptomeningeal dissemination from cerebral malignant gliomas without a recurrence of the primary lesions].

Reported are three cases of a spinal leptomeningeal dissemination from supratentorial malignant gliomas, in which the recurrence of the primary tumors of the intracranial leptomeningeal involvement was not observed clinicopathologically. All cases complained of spinal cord symptoms such as paraplegia or a sensory disturbance from time to time after the initial operation for their intracranial primary tumors. The duration between the first operation and the appearance of the spinal cord symptoms ranged from 11 to 17 months.

A case of immunoblastic lymphadenopathy (IBL)-like T-cell lymphoma with unusual clinical manifestations.

A 67-year-old male consulted an otolaryngologist with a complaint of dysphagia due to a large polypoid mass in the hypopharynx. A biopsy of the mass revealed immunoblastic lymphadenopathy (IBL)-like T-cell lymphoma of helper/inducer subset occurring in lymphoid tissues of the hypopharynx. Although general lymphadenopathy was not seen, further biopsy of an inguinal lymph node showed small foci of lymphoma cells, suggesting the generalized distribution of this malignancy.