Separation and analysis of charged isomers of monoclonal immunoglobulin G by ceramic hydroxyapatite chromatography.

Analysis of monoclonal antibody (MAb) heterogeneity caused by posttranslational modifications is important for pharmaceutical quality assurance of antibody drugs. In this study, by employing small ceramic hydroxyapatite (HAp) particles that were self-manufactured to have a 2.5-µm diameter, we attempted to separate and analyze MAb isomers. The MAb without N-linked oligosaccharides could be separated by 2.5-µm HAp chromatography as well as MAb with N-linked oligosaccharides. Hence, a variety of N-linked oligosaccharides do not appear to be involved in the separation mechanism of HAp chromatography. However, there is a difference in retention time between MAb with and without N-linked oligosaccharides, meaning that the presence of N-glycan could influence the retention time of HAp chromatography. Subsequently, the MAb fractions separated by 2.5-µm HAp chromatography were analyzed by isoelectric focusing, and seven isomers of the MAb having different isoelectric points (pI) were identified. The MAb isomers were eluted in order of lower pI isomers with sodium phosphate buffer, and enzyme-linked immunosorbent assay indicated the immunoreactivity of the fraction including the lowest pI isomers to be remarkably reduced. This study yielded details of the separation behavior of HAp chromatography owing to 2.5-µm HAp particles.

Sequential two-step chromatographic purification of infectious poliovirus using ceramic fluoroapatite and ceramic hydroxyapatite columns.

Infectious virus purification techniques are important for vaccine development and gene therapy applications. However, the standardized one-step purification technique using ceramic hydroxyapatite (CHAp) has proven unsuitable for poliovirus. Therefore, we designed a sequential two-step chromatographic technique for purification of the infectious Sabin type 2 vaccine strain of poliovirus from the cell culture supernatant. In the first step, we removed protein contaminants from the Sabin type 2 virus fraction by pH gradient elution on a ceramic fluoroapatite column. In the second step, we removed double-stranded DNA derived from host cells by diluting the virus fraction, directly loading it on a CHAp column, and purifying it using a phosphate gradient with 1 M sodium chloride. This process achieved removal rates of more than 99.95% and 99.99% for proteins and double-stranded DNA, respectively, and was highly reproducible and scalable. Furthermore, it is likely that it will be applicable to other virus species.

[External quality assessment of the genetic testings for hematopoietic tumor, CML].

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.

Scanning electron microscopy-based approach to understand the mechanism underlying the adhesion of dengue viruses on ceramic hydroxyapatite columns.

Although ceramic hydroxyapatite (HAp) chromatography has been used as an alternative method ultracentrifugation for the production of vaccines, the mechanism of virus separation is still obscure. In order to begin to understand the mechanisms of virus separation, HAp surfaces were observed by scanning electron microscopy after chromatography with dengue viruses. When these processes were performed without elution and with a 10-207 mM sodium phosphate buffer gradient elution, dengue viruses that were adsorbed to HAp were disproportionately located in the columns. However, when eluted with a 10-600 mM sodium phosphate buffer gradient, few viruses were observed on the HAp surface. After incubating the dengue viruses that were adsorbed on HAp beads at 37°C and 2°C, the sphericity of the dengue viruses were reduced with an increase in incubation temperature. These results suggested that dengue virus was adsorbed to the HAp surface by electronic interactions and could be eluted by high-salt concentration buffers, which are commonly used in protein purification. Furthermore, virus fusion was thought to occur with increasing temperature, which implied that virus-HAp adhesion was similar to virus-cell adhesion.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Surface chemical analysis and chromatographic characterization of polyethylenimine-coated hydroxyapatite with various amount of polyethylenimine.

Polyethylenimine (PEI) has been widely used as a coating material to produce stationary phase for ion-exchange chromatography of biomolecules. However, a precise study of the PEI coating fraction has been lacking, despite such quantification being very important for fundamental research as well as identifying further industrial applications. In this study, we produced four types of PEI-coated hydroxyapatite (PEI-HAp) with various fractions of PEI (0.16%, 0.5%, 1.0%, 1.5%) using a spray-drying system to evaluate correlations between coating fractions and the thermochemical or chromatographic behaviors of theses products. The thermal analyses of these matrices showed two exothermic peaks when the PEI coating fraction exceeded 1.0%. The one peak indicated a PEI decomposition peak and the other would indicate bond dissociation of PEI layers formed over the HAp surface as the PEI concentration increased. Furthermore, the chromatographic analysis for the surface chemical characteristics showed the correlation between coating fraction and the retention time of protein or nucleotide. Acidic or phosphorylated proteins were more strongly adsorbed as the PEI coating fraction increased when the initial coating fraction was low, but at fraction exceeding 0.5%, constant retention was observed. The retention time of nucleotides increased in proportion to the fraction of PEI added. The good selectivity of PEI-HAp may be attributable to multifunctional interactions of electrostatic and bare Ca sites on HAp, not just the amino sites of PEI. These precise studies of PEI coating fraction are our original novel contributions, which could be achieved by quantitative consideration using thermal analysis and chromatography.

A new time-resolved fluorometric microarray detection system using core-shell-type fluorescent nanosphere and its application to allergen microarray.

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab' fragment-conjugated fluorescent nanosphere (Fab' nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab' nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 mul of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r > 0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n = 5).