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Microfluidic Glycan Analysis

a microfluidic glycan and microfluidic technology, applied in the field of microfluidic glycan analysis, can solve the problems of large quantity of starting materials, time-consuming and cumbersome steps, and manual operation errors,

Inactive Publication Date: 2010-07-29
AGILENT TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new and improved approach to analyzing glycans, which are important components of cell surface and extracellular matrix proteins. The invention involves a microfluidic device that allows for faster, more efficient, and streamlined preparation, separation, and analysis of glycans compared to traditional methods. The device achieves higher recovery yields of glycans and superior chemical separation of isoforms with a significantly smaller amount of starting material required. The invention also provides a better understanding of the chemical mechanism of enzymatic action of PNGase F, which is involved in the deglycosylation process. Overall, the invention offers a more efficient and effective way to analyze glycans and their impact on disease progression and treatment.

Problems solved by technology

These steps are time-consuming and cumbersome, and the manual operations are error-prone.
Furthermore, a relatively large quantity of starting materials is required to generate high quality data.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Glycan Analysis with On-Chip Enzymatic Reactions

Chip Fabrication

[0069]PNGase F enzyme was chemically attached to epoxide-modified 5 μm silica beads in phosphate buffer (pH 8) over 22 hr. The beads were then packed into a chamber located in a first polyimide chip. Reverse-phase 5-μm C8 beads were packed into a second chamber on a second polyimide chip for protein cleaning. The third chip layer contained a sample enrichment column (40 nl), a separation column (75 mm) filled with graphitized carbon for HPLC analysis, and an integrated MS tip for input into a mass spectrometer. All three chips were stacked, aligned and inserted into a chip frame holder.

Methods

[0070]The antibody samples were diluted in phosphate buffer to a concentration of 6 pmol / μl in phosphate buffer (pH 7.5). The HPLC system operated using typical mobile phases of 0.1% formic acid in MS-grade water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). The autosampler was set to inject 0.1 μl of antibody. The ...

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Abstract

Microfluidic devices and methods for analyzing glycan profiles of glycoproteins are provided. Some embodiments of the devices comprise a deglycosylation column for cleaving glycans, an optional cleaning column for removing proteins, a trapping column for enriching glycans, and a separation column for resolving glycans. The devices and methods significantly improve the speed and sensitivity of glycan analysis.

Description

BACKGROUND OF THE INVENTION[0001]The analysis of glycoprotein structure and function, known as glycomics, has become a new and important area of research. Glycosylation is the most common post-translational modification of cell surface and extracellular matrix proteins. Glycoproteins play an important role in cell-adhesion and immune response. Changes in abundance and glycan profiles have been correlated with progression of diseases, such as cancer and rheumatoid arthritis. In addition, the analysis of glycan profiles is critical in the bio-therapeutic industry. For example, antibody bio-therapeutics contain glycosylated amino acids that assist in maintaining drug activity and preventing drug rejection by the immune system. Therefore, companies that make these bio-therapeutics have to monitor and verify their glycan profiles.[0002]Glycan analysis is usually performed by capillary electrophoresis or mass spectrometry methods. In either case, the glycans must be removed from the glyco...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N27/26C12M3/04C12M1/34C12Q1/00
CPCB01L3/502738H01J49/00B01L3/502761B01L2300/0874B01L2300/0887B01L2400/0487B01L2400/0644F16K99/0001G01N1/405G01N27/44791G01N30/465G01N30/6095G01N30/7266G01N33/6842G01N2030/085G01N2400/00B01L3/502753B01D15/08B01L2200/10B01L2300/0627B01L2400/0406B01L2400/0421
Inventor BYNUM, MAGDALENA A.GRIMM, RUDOLFKILLEEN, KEVIN P.ROBOTTI, KARIA M.
Owner AGILENT TECH INC