Effects of culture temperature and pH on flag-tagged COMP angiopoietin-1 (FCA1) production from recombinant CHO cells: FCA1 aggregation.

To maximize the production of flag-tagged cartilage oligomeric matrix protein angiopoietin-1 (FCA1) from Chinese hamster ovary (CHO) cells, the effects of culture pH and temperature on cell growth and FCA1 production were investigated. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37 °C). Lowering the culture temperature suppressed cell growth while allowing maintenance of high cell viability for a longer culture period. The specific FCA1 productivity (q (FCA1)) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33 °C, and was approximately 4.0-fold higher than that at pH 7.2 and 37 °C. However, aggregates and a monomeric form of FCA1, which are undesirable due to reduced biological activity or immunogenicity, were significant at pH 7.2 and 33 °C. It was also found that the expression pattern of FCA1 was affected more significantly by culture pH than by the culture temperature. FCA1 aggregation dramatically decreased at culture pH 7.5 regardless of the culture temperature. Furthermore, the monomeric form of FCA1 was not observed. Taken together, optimization of culture temperature and culture pH (33 °C and pH 7.5) significantly improves the production of biologically active FCA1 with tetrameric or pentameric forms from CHO cells.

Evaluation of Physicochemical and Biological Stability of 36-Months-Aged SB5 (Adalimumab Biosimilar) for 4 Weeks at Room Temperature.

INTRODUCTION: Tumor necrosis factor (TNF-alpha) inhibitors, such as adalimumab, have shown success in treating autoimmune inflammatory diseases but are associated with substantial financial burdens to the healthcare system. Biosimilars, which are highly similar to biologic agents, offer the potential to reduce the financial burden of treatment. In the case of TNF-alpha inhibitors, they may also offer improved stability and enable prolonged use. SB5, an adalimumab biosimilar, has shown equivalent efficacy and comparable safety to its reference product in clinical trials. Currently, SB5 is approved for storage for 36 months at 2-8 °C and may be stored at room temperature (25 °C) for a maximum period of 14 days. The objective of this study was to evaluate the stability of SB5, aged to its shelf-life of 36 months, at room temperature (25 ± 2 °C) and 60 ± 5% relative humidity (RH) for a period of 4 weeks, which is longer by 14 days than that of SB5 currently approved in the European Union. METHODS: This study evaluated the stability of SB5, aged to its shelf-life of 36 months, at room temperature (25 ± 2 °C) for a period of 4 weeks. Three independent batches of 36 months-aged SB5 were stored at 25 ± 2 °C and 60 ± 5% RH for 4 weeks. Samples were tested at 0, 2, and 4 weeks. RESULTS: Color, clarity, visible particles, pH, protein concentration, and particulate matter were consistent among the batches, and all the test results met the acceptance criteria at each time point. Percent charge variance was maintained over time. Percent of high molecular weight species detected, total purity, relative binding activity by TNF-alpha, and relative potency by TNF-alpha neutralization did not change over time within each batch, and all values were within the acceptance criteria limits. CONCLUSION: SB5 aged for 36 months is physicochemically and biologically stable for 4 weeks at 25 ± 2 °C and 60 ± 5% RH, which is 2 weeks longer than the alternative storage condition as approved by the European Medicines Agency, which is at 25 °C for a period of up to 14 days. FUNDING: Samsung Bioepis Co., Ltd.

Extended Stability of Reconstituted and Diluted SB3 (Trastuzumab Biosimilar) Assessed by Physicochemical and Biological Properties.

INTRODUCTION: Stability information for the trastuzumab biosimilar SB3 is limited to 48 h at 2-8 °C for the reconstituted solution and 24 h at up to 30 °C for diluted solutions. Extended physicochemical stability and biological activity were assessed to evaluate the advanced preparation of reconstituted and diluted SB3. METHODS: Under controlled and aseptic conditions, the stability of reconstituted and diluted SB3 was evaluated using several assessments and according to the UK's National Health Service guidance. Reconstituted SB3 was stored at 25 ± 2 °C with 60 ± 5% relative humidity for 3 days, and subsequently diluted SB3 (0.32-4 mg/mL) was stored in an infusion bag in the absence of light at 25 ± 2 °C with 60 ± 5% relative humidity for 28 days and 5 ± 3 °C for 28 days, respectively. Physicochemical stability (appearance, pH, protein concentration, size exclusion high-performance liquid chromatography, non-reducing capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (competitive inhibition binding assay to human epidermal growth factor receptor 2 by fluorescence resonance energy transfer, anti-proliferation assay), and properties with a potential safety impact (subvisible particulates, submicronic aggregation by dynamic light scattering) were determined. RESULTS: No physicochemical instability signs or biological activity changes were observed for either reconstituted or diluted SB3 up to 28 days; all stability acceptance criteria were met. No major change was noted in the proportion of molecular weight variants (high molecular weight impurity, total purity) or relative percentages of acidic, main, and basic charge isoforms of the protein. No increases in particulates or aggregates in terms of a potential safety impact were noted. CONCLUSION: The physicochemical stability and biological activity of reconstituted and diluted SB3 are maintained for extended time periods beyond those denoted in the product labeling, which allows for advanced SB3 preparation and may reduce drug wastage and preparation time. FUNDING: Samsung Bioepis Co., Ltd.

Magnetoencephalography as a Prognostic Tool in Patients with Medically Intractable Temporal Lobe Epilepsy.

BACKGROUND: Most surgical treatments for medically intractable temporal lobe epilepsy are helpful. When a patient has persistent symptoms after surgery, there are no tests that accurately predict whether a patient will have remnant epileptic foci. The aim of this study was to evaluate the usefulness of magnetoencephalography (MEG) as a prognostic tool in patients with temporal lobe epilepsy. METHODS: From July 2012 to July 2016, 21 patients underwent preoperative and postoperative MEG at our center. Postoperative MEG was performed within 2 weeks after surgery. We analyzed MEG by estimating the time-frequency component of the signal to define gamma oscillations (GOs), which are an indicator of epileptogenic foci. We analyzed the relationship between GOs on MEG and surgical outcomes. RESULTS: Mean follow-up period was 28.3 months (range, 13-44 months). At the last follow-up visit, patients were divided into 2 groups according to surgical outcome. All patients showed spike waves and GOs on preoperative electroencephalography and MEG. In the seizure control group (16 patients), spike waves (2 patients) and GOs (2 patients) were seen postoperatively despite absence of symptoms. In the recurrent seizure group (5 patients), whereas 3 patients showed spike waves, all 5 patients showed GOs on MEG postoperatively. There was a significant association between presence of GOs on postoperative MEG and surgical outcome (P = 0.01). CONCLUSIONS: MEG can provide valuable postsurgical information on epileptic foci in patients with recurrent symptoms; GOs on postoperative MEG were especially correlated with epileptic recurrence. Our data show that GOs on postoperative MEG may have prognostic value.

High-level expression and purification of a designed angiopoietin-1 chimeric protein, COMP-Ang1, produced in Chinese hamster ovary cells.

A designed angiopoietin-1 (Ang1) chimeric protein with nonleaky angiogenic activity, COMP-Ang1, is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (>20 microg/mL) of COMP-Ang1 and an amino-terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, 0.32, and 1 microM. The COMP-Ang1 secreted from rCHO cells was purified at a purification yield of 40.3% from the culture medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secrete COMP-Ang1 in homopentameric and homotetrameric glycoprotein forms. Furthermore, COMP-Ang1 binds to the Tie2 receptor and phosphorylates Tie2, indicating its potential for therapeutic angiogenesis.

Symptom-associated change of motor-related neuromagnetic fields in a patient with multiple sclerosis: A case report.

The objective of this study was to investigate functional abnormalities of the brain in a patient with multiple sclerosis (MS) by using magnetoencephalography (MEG) and a finger-tapping task. A 46-year-old woman that presented with motor weakness of left hand and was diagnosed with MS. Conventional magnetic resonance imaging demonstrated a white matter lesion with hyperintensity on T2-weighted images in the right motor area. MEG recordings were performed during the period of motor weakness and after clinical improvement. Neuromagnetic brain activation was elicited by a simple, visually cued finger movement. The Equivalent current dipole (ECD) strength of the movement-evoked field (MEF) in the affected hemisphere was significantly decreased relative to the unaffected hemisphere. After improvement in motor weakness, we found that the lower amplitude of the readiness field and decreased ECD strength of the MEF observed in affected hemisphere during motor weakness had recovered. Analysis of motor-related neuromagnetic fields revealed that MEG may be used to detect diffuse changes in the brain that are not observable by conventional imaging of white matter regions in MS. We further found that brain activities can change after improvement in motor weakness.

Expression and purification of recombinant human angiopoietin-1 produced in Chinese hamster ovary cells.

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 microM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.

Current issues and areas for improvement in the Korean Dental Hygienist National Licensing Examination: an expert Delphi survey among dental hygienists.

PURPOSE: This study aimed to investigate current issues and areas for improvement in the Korean Dental Hygienist National Licensing Examination (KDHNLE) through an expert Delphi survey. METHODS: A Delphi survey was conducted from May through August 2016 in Korea. This Delphi survey included 20 persons representing the field of dental hygiene (7 groups from various dental hygiene-related organizations). The Delphi survey was administered through e-mail as 3 rounds of questionnaire surveys regarding the issues facing the KDHNLE and potential solutions to those challenges. The primary Delphi survey was an open questionnaire. In each round, subjects' responses were categorized according to the detailed themes of their responses. The minimum value of the content validity ratio of the survey results was determined by the number of panels participating in the Delphi survey. RESULTS: Issues facing the KDHNLE were identified from the results of the Delphi survey. The following 4 items had an average importance score of 4.0 or higher and were considered as important by over 85% of the panels: the failure of the practical test to reflect actual clinical settings, the focus of the practical test on dental scaling, the gap between the items evaluated on the national examination and actual practical work, and insufficiency in strengthening the expertise of licensed dental hygienists. The following items were suggested for improvement: more rigorous rater training, adjustment of the difficulty of the licensing examination, the introduction of a specialized dental hygienist system, and more rigorous refresher training for licensed dental hygienists. CONCLUSION: Based on the above results, the KDHNLE should be improved according to the core competencies of dental hygienists, including on-site clinical practice experience.

Molecular epidemiology of norovirus in asymptomatic food handlers in Busan, Korea, and emergence of genotype GII.17.

The molecular epidemiology of norovirus infections was studied in food handlers without any symptoms from January to December 2015 in Busan city, Korea. A total of 2,174 fecal specimens from asymptomatic food handlers were analyzed, and 2.3% (49/2,174) were norovirus-positive. Fourteen of 335 samples (4.2%) were positive in January; fifteen of 299 samples (5.0%) in February, and seven of 189 samples (3.7%) in December. However, norovirus was rarely detected in other months. From sequencing analysis, 11 genotypes (five GI and six GII genotypes) were detected. Among the 42 capid gene sequences identified, 14 were from the GI genogroup, while 28 were from the GII genogroup. The most commonly detected genotype was GII.17, comprising 15 (35.7%) of positive samples. From January 2012 to December 2015, 5,138 samples were collected from gastroenteritis patients and outbreaks in Busan. The most detected genotype in 2012, 2013, and 2014 was GII.4 (121, 24, and 12 cases, respectively), but in 2015, GII.17 (25 cases) was the most common. The GII.4 genotype was the major cause of acute gastroenteritis from 2012 to 2014, but the GII.17 genotype became the most prevalent cause in 2015. Continued epidemiological surveillance of GII.17 is needed, together with assessment of the risk of norovirus infection.

Biological characterization of angiopoietin-3 and angiopoietin-4.

The angiopoietin (Ang) family of growth factors includes Ang1, Ang2, Ang3, and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang3 (mouse) and Ang4 (human) are interspecies orthologs. In experiments with human endothelial cell lines, Ang3 was identified as an antagonist of Tie2 and Ang4 was identified as an agonist of Tie2. However, the biological roles of Ang3 and Ang4 are unknown. We examined the biological effect of recombinant Ang3 and Ang4 proteins in primary cultured endothelial cells and in vivo in mice. Recombinant Ang3 and Ang4 formed disulfide-linked dimers. Ang4 (400 ng/mL) markedly increased Tie2 and Akt phosphorylation in primary cultured HUVECs whereas Ang3 (400 ng/mL) did not produce significant changes. Accordingly, Ang4, but not Ang3, induced survival and migration in primary cultured HUVECs. Unexpectedly, intravenously administered Ang3 (30 microg) was more potent than Ang4 (30 microg) in phosphorylating the Tie2 receptor in lung tissue from mice in vivo. Accordingly, Ang3 was more potent than Ang4 in phosphorylating Akt in primary cultured mouse lung microvascular endothelial cells. Ang3 and Ang4 both produced potent corneal angiogenesis extending from the limbus across the mouse cornea in vivo. Thus, Ang3 and Ang4 are agonists of Tie2, but mouse Ang3 has strong activity only on endothelial cells of its own species.

Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production.

Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells.

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.

Use of NaCl prevents aggregation of recombinant COMP-angiopoietin-1 in Chinese hamster ovary cells.

To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)-Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300-450mOsm/kg. The specific productivity of COMP-Ang1, q(COMP-Ang1), increased as medium osmolality increased. At NaCl-450mOsm/kg, the q(COMP-Ang1) was 7.7-fold higher than that at NaCl-300mOsm/kg, while, at sorbitol-450mOsm/kg, it was 2.9-fold higher than that at sorbitol-300mOsm/kg. This can be attributed to the increased relative mRNA level of COMP-Ang1 at NaCl-450mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450mOsm/kg. Western blot analysis showed that COMP-Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP-Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP-Ang1 was drastically reduced at NaCl-400mOsm/kg. At NaCl-450mOsm/kg, the aggregation of COMP-Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP-Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP-Ang1 production and reducing COMP-Ang1 aggregation.

Artificial transcription factors increase production of recombinant antibodies in Chinese hamster ovary cells.

A randomized library that encodes for artificial zinc finger protein transcription factors (ZFP-TF) was constructed and screened for components that increased production of a monoclonal antibody (mAb-72) in Chinese hamster ovary (CHO) cells. One of these ZFP-TF, LK52, increased mAb-72 production approximately 10-fold at approximately 60% transduction efficiency; a mutated version of LK52, however, did not boost mAb-72 production. LK52 also increased production of other mAbs in CHO cells. These results demonstrate that ZFP-TF libraries can be used to identify components that improve antibody production in CHO cells.

Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin.

Sodium butyrate (NaBu) can enhance the expression of foreign protein of recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on foreign protein expression in rCHO cells is compromised by its growth inhibitory and cytotoxic effects. To overcome this cytotoxic effect of NaBu, an expression vector of small interfering RNAs (siRNAs) targeting against caspase-3, a key effector component in apoptosis, was constructed and transfected into rCHO cells producing human thrombopoietin (hTPO). Using this siRNA strategy, rCHO cells (F21 cells) expressing a low level of caspase-3 proenzyme determined by RT-PCR and Western blot analysis were established. Under the condition of 1-5 mM NaBu addition at the exponential growth phase, down-regulation of caspase-3 in F21 cells could not effectively inhibit NaBu-induced apoptotic cell death. This NaBu-induced apoptotic cell death occurred because F21 cells appeared to compensate for the lack of caspase-3 by increasing the active caspase-7 level. These results suggest that the intracellular caspase's interconnectivity should be taken into consideration for the successful inhibition of apoptosis of rCHO cells.

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells.

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.

Two juvenile hormone suppressible storage proteins may play different roles in Hyphantria cunea Drury.

We isolated and sequenced cDNA clones corresponding to two storage proteins (HcSP-1 and HcSP-2) from fall webworm, Hyphantria cunea. The cDNAs for HcSP-1 (2,337 bp) and HcSP-2 (2,572 bp) code for 753 and 747 residue proteins with predicted molecular masses of 88.3 and 88.5 kDa, respectively. The calculated isoelectric points are pI = 8.4 (HcSP-1) and 7.6 (HcSP-2). Multiple alignment analysis of the amino acid sequence revealed that HcSP-1 is most similar to SL-1 from S. litura (73.8% identity) and other methionine-rich hexamers, whereas HcSP-2 is most similar to the SL-2 alpha subunit from S. litura (74.8% identity) and other moderately methionine-rich hexamers. The two storage proteins from H. cunea shared only 38.4% identity with one another. According to both phylogenetic analyses and the criteria of amino acid composition, HcSP-1 belongs to the subfamily of Met-rich storage proteins (6% methionine, 10% aromatic amino acid), and HcSP-2 belongs to the subfamily of moderately methionine-rich storage proteins (3.2% methionine, 12.9% aromatic amino acid). Topical application of the JH analog, methoprene, after head ligation of larvae, suppressed transcription of the SP genes, indicating hormonal effects at the transcriptional level. The HcSP-1 transcript was detected by Northern blot analysis in Malpighian tubule, testis, and ovary, in addition to fat body where it was most abundant. The HcSP-2 transcript was detected only in fat body and Malpighian tubule. The accumulation of HcSP-1 in ovary and HcSP-2 in Malpighian tubule might be related to differential functions in both organs.