Olajos M.,Debrecen University |
Olajos M.,University of Pannonia |
Szekrenyes A.,Debrecen University |
Hajos P.,University of Pannonia |
And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010
In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5-65 °C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 °C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 °C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate. © 2010 Springer-Verlag. Source
Fritz J.S.,Iowa State University |
Gjerde D.T.,PhyNexus Inc
Journal of Chromatographic Science | Year: 2010
This year marks the 30th anniversary of the publication of Non-Suppressed Ion Chromatography, which is a method for the rapid separation of anions with on-line conductimetric detection. In this method, the separation column is connected directly to the conductimetric detector. This single-column method is a simpler technique than the original suppressed ion chromatography method, which requires a large suppressor column to reduce the background conductance. In the new method, the background signal is reduced to a manageable level simply by using an ionexchange separation column of low exchange capacity that lowers the eluent concentration needed for separation. The eluent ion used for separation is chosen based on having large, bulky structure, which lowers the equivalent conductance and facilitates detection of the sample anions. This is a personal account of the initial discovery and early development of non-suppressed ion chromatography. The circumstances for the discovery are recounted by the two authors. Methods are described for determination of anions, cations with indirect detection, and techniques for increasing detection sensitivity. A fundamental equation for the prediction of ion chromatography detector response is given, and the development of several types of detection schemes for ion chromatography is discussed. Finally, the impact of non-suppressed ion chromatography is discussed together with comments on the discovery process. Source
Szigeti M.,Debrecen University |
Bondar J.,University of Pannonia |
Gjerde D.,PhyNexus Inc |
Keresztessy Z.,Debrecen University |
And 3 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2016
N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10. min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots. © 2016. Source
PhyNexus Inc. | Date: 2008-03-25
Biotechnology analysis instrumentation, supplies, and technology for the analysis of proteins, polynucleotides, polysaccharides, lipids and other biomolecules; namely, absorptive separation devices in the nature of laboratory pipettes, columns and capillaries.
PhyNexus Inc. | Date: 2012-05-01
Chemicals, namely, buffer and standard solutions used in analytical chemistry; Kit containing pre-packed columns, chemicals, pre-made buffer concentrates, syringes and instructional manual for the purification of proteins for in vitro use. Laboratory apparatus and instruments, namely, pre-packed columns for use in separation and purification; Scientific apparatus and instruments, namely, fluid handling device used for disposable bioprocessing applications and parts and fittings therefor.