• FOCUS: LIFE SCIENCE
• /
• 제1호
• /
• pp.3.1-3.7
• /
• 2024

The article discusses the critical role of chromatography in the analysis and purification of proteins in biopharmaceuticals, emphasizing the importance of comprehensive characterization for ensuring their safety and efficacy. It highlights the use of Avantor® ACE® HPLC columns for the separation and purification of proteins, focusing on the analysis of intact proteins using reversed-phase liquid chromatography (RPLC) with fully porous particles. This article also details the application of different mobile phase additives, such as TFA and formic acid, and emphasizes the advantages of using type B ultra-pure silica-based columns for efficiency and peak shape in biomolecule analysis. Additionally, it addresses the challenges of analyzing intact proteins due to slow molecular diffusion and introduces the concept of solid-core (or superficially porous) particles, emphasizing their benefits over traditional porous particles for the analysis of therapeutic proteins. Furthermore, it discusses the development of Avantor® ACE® UltraCore BIO columns, specifically designed for the high-efficiency separation of large biomolecules, such as proteins, and demonstrates their effectiveness in achieving high-resolution separations, even for higher molecular weight proteins like monoclonal antibodies (mAbs). In addition, it underscores the complexity of analyzing and characterizing intact protein biopharmaceuticals, requiring a range of analytical techniques and the use of wide-pore stationary phases, operated at elevated temperatures and with relatively shallow gradients. It highlights the comprehensive range of options offered by Avantor® ACE® wide pore columns, including both fully porous and solid-core particles, bonded with a variety of complementary stationary phase chemistries to optimize selectivity during method development. The use of ultrapure and highly inert base silica is emphasized for enabling the use of lower concentrations of mobile phase modifiers without compromising analyte peak shape, particularly beneficial for LC-MS applications. Then the article concludes by emphasizing the significance of reversed-phase liquid chromatography and its compatibility with mass spectrometry as a valuable tool for the separation and analysis of intact proteins and their closely related variants in biopharmaceuticals.

• 대한전기학회:학술대회논문집
• /
• 대한전기학회 2004년도 하계학술대회 논문집 C
• /
• pp.2102-2104
• /
• 2004

In this study, sheathless electrospray from PDMS/glass microchips with conducting metal emitter tip is described. A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system has advantages of the CE separation and on-line electrospray detection of peptide solution. We have fabricated a new electrospray ionization(ESI) device composed of the metal emitter tip and CE separation channel monolithically in a glass microchip. The separation channel and metal emitter tip are fabricated using a glass wet etching and gold electro plating process, respectively. The fabricated micro electrospray chip was tested by spraying peptide sample for mass spectrometric analysis. Singlely-charged peak and doublely-charged peak of peptide were detected and further MS/MS fragmentation was performed in each peak. Direct comparisons with conventional glass or fused silica emitters showed very similar performance with respect to signal strength and stability.

• BMB Reports
• /
• 제45권4호
• /
• pp.233-238
• /
• 2012

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.

• Korean Membrane Journal
• /
• 제6권1호
• /
• pp.16-23
• /
• 2004

The silica containing carbon (C-SiO$_2$) membranes were fabricated using poly(imide siloxane) (PIS) having -CO- swivel group. The characteristics of porous C-SiO$_2$ structures prepared by the pyrolysis of poly(imide siloxane) were related with the micro-phase separation between the imide block and the siloxane block. Furthermore, the nitrogen adsorption isotherms of the CMS and the C-SiO$_2$ membranes were investigated to define the characteristics of porous structures. The C-SiO$_2$ membranes derived from PIS showed the type IV isotherm and possessed the hysteresis loop, which was associated with the mesoporous carbon structures, while the CMS membranes derived from PI showed the type I isotherm. For the molecular sieving probe, the C-SiO$_2$ membranes pyrolyzed at 550, 600, and 700$^{\circ}C$ showed the O$_2$ permeability of 924, 1076, and 367 Barrer (1 ${\times}$ 10$\^$-10/㎤(STP)cm/$\textrm{cm}^2$$.$s$.$cmHg) and O$_2$/N$_2$ selectivity of 9, 8, and 12.

• ETRI Journal
• /
• 제34권2호
• /
• pp.226-234
• /
• 2012

The novelty of this study resides in the fabrication of stoichiometric and stress-reduced $Si_3N_4/SiO_2/Si_3N_4$ triple-layer membrane sieves. The membrane sieves were designed to be very flat and thin, mechanically stress-reduced, and stable in their electrical and chemical properties. All insulating materials are deposited stoichiometrically by a low-pressure chemical vapor deposition system. The membranes with a thickness of 0.4 ${\mu}m$ have pores with a diameter of about 1 ${\mu}m$. The device is fabricated on a 6" silicon wafer with the semiconductor processes. We utilized the membrane sieves for plasma separations from human whole blood. To enhance the separation ability of blood plasma, an agarose gel matrix was attached to the membrane sieves. We could separate about 1 ${\mu}L$ of blood plasma from 5 ${\mu}L$ of human whole blood. Our device can be used in the cell-based biosensors or analysis systems in analytical chemistry.

• Archives of Pharmacal Research
• /
• 제27권10호
• /
• pp.1009-1015
• /
• 2004

Quantitative Structure-Resolution Relationship (QSRR) using the Comparative Molecular Field Analysis (CoMFA) software was applied to predict the chromatographic behaviors of chiral drugs with an amine moiety on the chiral cellobiohydrolase (CBH) columns. As a result of the Quantitative CoMFA-Resolution Relationship study, using the partial least square method, prediction of the behavior of drugs with amine moiety upon chiral separation became possible from their three dimensional molecular structures. When a mixed mobile phase of 10 mM aqueous phosphate buffer (pH 7.0) - isopropanol (95 : 5) was employed, the best Quantitative CoMFA-Resolution Relationship, derived from the study, provided a cross-validated $q^2$ = 0.933, a normal $r^2$ = 0.995, while the best Quantitative CoMFA-Separation Factor Relationship, also derived from the study, yielded a cross-validated $q^2$ = 0.939, a normal $r^2$ = 0.991. When all of these results are considered, this QSRR-CoMFA analysis appears to be a very useful tool for the preliminary prediction on the chromatographic behaviors of drugs with an amine moiety inside chiral CBH columns.

• Bulletin of the Korean Chemical Society
• /
• 제16권9호
• /
• pp.873-885
• /
• 1995

The local structural and thermodynamical properties of blends A-B/H of a diblock copolymer A-B and a homopolymer H are studied using the polymer reference interaction site model (RISM) integral equation theory with the mean-spherical approximation closure. The random phase approximation (RPA)-like static scattering function is derived and the interaction parameter is obtained to investigate the phase transition behaviors in A-B/H blends effectively. The dependences of the microscopic interaction parameter and the macrophase-microphase separation on temperature, molecular weight, block composition and segment size ratio of the diblock copolymer, density, and concentration of the added homopolymer, are investigated numerically within the framework of Gaussian chain statistics. The numerical calculations of site-site interchain pair correlation functions are performed to see the local structures for the model blends. The calculated phase diagrams for A-B/H blends from the polymer RISM theory are compared with results by the RPA model and transmission electron microscopy (TEM). Our extended formal version shows the different feature from RPA in the microscopic phase separation behavior, but shows the consistency with TEM qualitatively. Scaling relationships of scattering peak, interaction parameter, and temperature at the microphase separation are obtained for the molecular weight of diblock copolymer. They are compared with the recent data by small-angle neutron scattering measurements.

• Macromolecular Research
• /
• 제10권1호
• /
• pp.18-23
• /
• 2002

We theoretically consider blends of two monodisperse one-end-functionalized homopolymers (denoted by A and B) capable of forming clusters between functional groups (stickers) using weak segregation theory. In this model system resulting molecular architectures via clustering resemble star copolymers having many A- and B-arms. Minimizing the total free energy with respect the cluster distribution, the equilibrium distribution of clusters is obtained and used for RPA (Random Phase Approximation) equations as input. For the case that polymers are functionalized by only one kind of sticker, the phase diagrams show that the associations promote the macrophase separation. When there is strong affinity between stickers belonging to the different polymer species, on the other hand, the phase diagram show a suppression of the macrophase separation at the range of high temperature regime, as well as the phase coexistence between a disordered and a mesoscopic phase at the relatively lower temperatures.

• Korean Chemical Engineering Research
• /
• 제43권2호
• /
• pp.266-271
• /
• 2005

혼합유기산으로부터 숙신산의 분리를 단극 분리막을 사용한 탈염전기투석장치를 이용하여 행하였다. 전류효율, 회수율, 에너지소요량, 분리비 등의 변화를 pH 값과 초기농도비에 따라 측정하였다. 숙신산의 분리는 숙신산과 아세트산의 이온화율, 분자량, 이온전도도, 전자가 크기 그리고 초기농도비 등으로 해석될 수 있었다.

• 한국대기환경학회지
• /
• 제9권1호
• /
• pp.101-106
• /
• 1993

In order to separate the Freon-12 and air mixture$(CF_2Cl_2/Air=0.1/99.9 vol.%)$ by pressure swing adsorption (PSA), the breakthrough curve was experimentally observed in a fixed bed adsorption column. A single adsorber was packed with various adsorbents such as, the activated carbon(S-AC, W-AC) and the molecular sieve(MS-5A, MS-13X). The order of appearance of breakthrough curve is MS-5A > MS-13X > W-AC > S-AC. The activated carbon was found to be more effective adsorbent for separating Freon-12 from the mixture than the molecular sieve was. From the experimental data obtained by the separation of Freon-12 gas out of the air stream in the steady-state PSA process cycle, whose size is the same one of column used for the breakthrough curve observation, it has been confirmed that Freon-rich gas could be obtained from the purge step of PSA and Freon-free air could be obtained from the adsorption step of PSA cycle.