• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.510-513
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• 2002

Iron is an essential nutrient for most organisms, which supplied to them in a protein-iron complex known as ferritin. Ferritins are multimeric proteins those are consisted of spherical shell of 24 subunits defining a cavity of about 8nm in diameter. Soluble form of ferritin was separated from disrupted cells, followed by silica powder adsorption. Ferritin was recovered from silica-poweder by distiiled water, which was applied to DEAE anion exchage chromatography. Collected fractions from the DEAE column were assayed to gain the amount and the purity of ferritin by using GF-HPLC.

• Korean Journal of Food Science and Technology
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• v.31 no.5
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• pp.1378-1385
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• 1999

The antioxidative activity of Fermented Anchovy on linoleic acid autooxidation was investigated in an aqueous system at pH 7.0. All solvent fractions from Fermented Anchovy were exhibited the strong antioxidative activity. Especially, BuOH and aqueous fractions were gained large amounts with strong antioxidative activity. Ultrafiltration, dialysis, heat treatment of aqueous fraction indicated that water-soluble antioxidants of Fermented Anchovy were heat-resistant, amino acid related compounds with smaller molecular weights than 1,000. Unbound fractions from DE-52 anion exchange chromatography were exhibited antioxidative activity with or without $15\;{\mu}M\;Fe^{+++}\;ion$. We were able to purify one methionine derivative from lots of antioxidative substances in Fermented Anchovy aqueous fraction by gel filtration, anion-exchange chromatography, TLC and HPLC, successfully. These data suggest that Fermented Anchovy aqueous fraction is a mixture of fermented small molecules with strong antioxidative activities.

• Korean Journal of Food Science and Technology
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• v.35 no.5
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• pp.791-796
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• 2003

Polyphenol oxidase isoforms were purified from the lotus roots using 50% acetone precipitation, conventional chromatographies of Q-Sepharose and hydrophobic interaction, and high performance liquid chromatographies of Mono-Q and Superdex 75 gel-filtration. Molecular mass of a purified PPO isoform (LPIII-2) was determined to be 56 kDa using gel-filtration chromatography. The active form of LPIII-2 appeared to bea heterodimer, as purified LPIII-2 on SDS-PAGE gel showed two bands that were determined to be 28 kDa and 26 kDa. To further characterize PPO, partially purified PPO isoforms (LP-II, LP-III) were obtained from Q-Sepharose anion-exchange chromatography. In substrate specificity, the partially purified PPO isoform LP-II showed a high affinity to catechol, while LP-III showed a high affinity to pyrogallol. The optimum pH of LP-II and LP-III was pH 7.0. Interestingly, the partially purified PPO isoforms showed high activities at low temperatures $(0{\sim}5^{\circ}C)$, and as temperatures rose, the activities decreased. Both PPO isoforms were stable at $40^{\circ}C$ and were inactivated by incubation at $60^{\circ}C$ for 40 min.

• Analytical Science and Technology
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• v.25 no.6
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• pp.483-491
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• 2012

This study is to investigate the issues on how to secure stability during the purification process for the production of recombinant protein A. The final recombinant protein A is produced by passing through the cation exchange column (SP) and the anion-exchange column (Q) during the production process, for which the samples produced by the step-by-step processes can be exposed to trouble in securing stable storage in case the next process cannot be taken within the proper time period. Accordingly, this study aims to evaluate the proper storage conditions and length of time when storing samples produced in the production process. That is, in this study, how to store fair samples, how long the storage period should be set up, and how to evaluate the security of its quality depending on time are dealt with. The items to be experimented with were enodotoxin, SDS-PAGE, HPLC purity and concentration. Experimental results showed that after passing the cation exchange column, when stored at $4^{\circ}C$ or room temperature, SDS-PAGE showed a major band, endotoxin is 5.0 Eu/mg or less, and concentration is on average of 8.21 to 8.24 mg/mL and RSD% 0.10~0.62%. In addition, HLPC purity showed somewhat stable results; at the HPLC purity 214 nm, the average is 99.24% to 99.37% and RSD% is 0.22~0.29%, while the average is 89.72% to 89.80% and RSD% 0.62~1.26% at 280 nm. On the contrary, after passing the anion exchange column, when stored at $4^{\circ}C$ or room temperature, SDS-PAGE revealed the major band, endotoxin is 0.5 Eu/mg or less, and concentration is on average of 5.59 mg/mL and RSD% 0.03~0.10%. when it comes to HLPC purity, the result showed that at the HPLC purity 214 nm, the average is 99.74% and RSD% is 0.10~0.11%, while the average is 96.16% to 96.85% and RSD% 0.72~1.13%. In conclusion, the stability of fair samples of recombinant protein A during the manufacturing process could be obtained without substance decomposition for 7~8 days at $4^{\circ}C$ or 20~21 days at room temperature.

• Bulletin of the Korean Chemical Society
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• v.34 no.12
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• pp.3817-3824
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• 2013

Various separation modes in HPLC, such as anion exchange (AE), reversed-phase (RP), and affinity (AF) chromatography were examined for the separation of selenium species in human blood serum and urine. While RP- and AE-HPLC were mainly used for the separation of small molecular selenium species, double column AF-HPLC achieved the separation of selenoproteins in blood serum efficiently. Further, the effluent of AF-HPLC was enzymatically hydrolyzed and then analyzed with RP HPLC for selenoamino acid study. The versatility of the hybrid technique makes the in-depth study of selenium species possible. For quantification, post column isotope dilution (ID) with $^{78}Se$ spike was performed. ORC ICP/MS (octapole reaction cell inductively coupled plasma/mass spectrometry) was used with 4 mL $min^{-1}$ Hydrogen as reaction gas. In urine sample, inorganic selenium and SeCys were identified. In blood serum, selenoproteins GPx, SelP and SeAlb were detected and quantified. The concentration for GPx, SelP and SeAlb was $22.8{\pm}3.4\;ng\;g^{-1}$, $45.2{\pm}1.7\;ng\;g^{-1}$, and $16.1{\pm}2.2\;ng\;g^{-1}$, respectively when $^{80}Se/^{78}Se$ was used. The sum of these selenoproteins ($84.1{\pm}4.4\;ng\;g^{-1}$) agrees well with the total selenium concentration measured with the ID method of $87.0{\pm}3.0\;ng\;g^{-1}$. Enzymatic hydrolysis of each selenium proteins revealed that SeCys is the major amino acid for all three proteins and SeMet is contained in SeAlb only.

• FOCUS: LIFE SCIENCE
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• no.1
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• pp.1.1-1.4
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• 2024

This article provides comprehensive guidance on the maintenance, cleaning, regeneration, and storage of silica-based HPLC (High-Performance Liquid Chromatography) columns. The general considerations emphasize the importance of using in-line filters and guard cartridges to protect columns from blockage and irreversible sample adsorption. While these measures help, contamination by strongly adsorbed sample components can still occur over time, leading to an increase in back pressure, loss of efficiency, and other issues. To maximize column lifetime, especially with UHPLC (Ultra-High Performance Liquid Chromatography) columns, it is advisable to use ultra-pure solvents, freshly prepared aqueous mobile phases, and to filter all samples, standards, and mobile phases. Additionally, an in-line filter system and sample clean-up on dirty samples are recommended. However, in cases of irreversible compound adsorption or column voiding, regeneration may not be possible. The document also provides specific recommendations for column cleaning procedures, including the flushing procedures for various types of columns such as reversed phase, unbonded silica, bonded normal phase, anion exchange, cation exchange, and size exclusion columns for proteins. The flushing procedures involve using specific solvents in a series to clean and regenerate the columns. It is emphasized that the flow rate during flushing should not exceed the specified limit for the particular column, and the last solvent used should be compatible with the mobile phase. Furthermore, the article outlines the storage conditions for silica based HPLC columns, highlighting the impact of storage conditions on the column's lifetime. It is recommended to flush all buffers, salts, and ion-pairing reagents from the column before storage. The storage solvent should ideally match the one used in the initial column test chromatogram provided by the manufacturer, and column end plugs should be fitted to prevent solvent evaporation and drying out of the packing bed.

• Bulletin of the Korean Chemical Society
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• v.34 no.9
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• pp.2663-2670
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• 2013

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature ($25^{\circ}C$ and $40^{\circ}C$) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at $40^{\circ}C$ and pH 9, respectively.

• Food Science and Biotechnology
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• v.18 no.4
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• pp.872-877
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• 2009

By-products of marine organisms including salmon, skate, flatfish, and yellow goosefish were investigated to search for new source of chondroitin sulfate (CS). Agarose gel electrophoresis with chondroitinase depolymerization showed that purified chondroitin sulfate did not contain any other glycosaminoglycans. 1H-nuclear magnetic resonance (NMR) spectra were acquired to confirm the structure and purity. The average molecular weight ranging from 22 to 64 kDa was determined by high performance size exclusion chromatography. Disaccharide compositions and purities were determined by strong anion exchange-high performance liquid chromatography (SAX-HPLC) after chondroitinase ABC depolymerization. SAX-HPLC data exhibited that the purity was from $81.7{\pm}1.3$ to $114.2{\pm}2.5%$ and the yield was from 1.3 to 12.5%. All analytical results indicate that salmon cartilage, skate cartilage, and yellow goosefish bone could be promising sources of CS to substitute shark cartilage CS in commercial neutraceuticals.

• Archives of Pharmacal Research
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• v.21 no.5
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• pp.555-558
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• 1998

chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. by the action of chondroitinase ABC, three unsaturated disaccharides$ 2-acetamide-2-deoxy-3-O-({\beta}-D-gluco-4-enepyranosyluronic acid)-D-galactose $$({\Delta}Di-OS), $2-acetamide-2-deoxy-3-O-({\beta}-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose ({\Delta}Di-6S) and 2-acetamide-2-deoxy-3-O-({\beta}-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose ({\Delta}Di-4S)$ were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of ${\Delta}Di-OS/{\Delta}Di-6S/{\Delta}Di-4S$ was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1789-1791
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• 2008

This study was aimed (i) to develop an effective method for the purification of ginsenosides for industrial use and (ii) to compare the distribution of ginsenosides in cultured wild ginseng roots (adventitious root culture of Panax ginseng) with those of red ginseng (steamed ginseng) and white ginseng (air-dried ginseng). The crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng were obtained by using a 75% ethanol extraction combined with ultrasonication. This was followed sequentially by AB-8 macroporous adsorption chromatography, Amberlite IRA 900 Cl anion-exchange chromatography, and Amberlite XAD16 adsorption chromatography for further purification. The contents of total ginsenosides were increased from 4.1%, 12.1%, and 11.3% in the crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng to 79.4%, 71.7%, and 72.5% in the final products, respectively. HPLC analysis demonstrated that ginsenosides in cultured wild ginseng roots were distributed in a different ratio compared with red ginseng and white ginseng.