Rapid and accurate analysis of protein sialylation
Inventors
Wang, Zihao • Sloan, Jessica • Wee, Kevin
Assignees
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Publication Number
US-9260743-B2
Publication Date
2016-02-16
Expiration Date
Abstract
The invention relates to methods and kits for the analysis of the sialylation of gluco-proteins. The samples of gluco-protein are incubated separately under three conditions: with beta-galactosidase, with beta-galactosidase+alpha-sialidase, and without an enzyme. After the enzyme treatment, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC PAD) is used to make a quantitative determination of the total galactose in the sample, the non sialylated galactose and the exogenous galactose in the medium. The determination of said values makes it possible to deduce the percentage of sialylation of the gluco-protein.
Core Innovation
The disclosure describes a method for determining the percentage of capped galactose in a glycoprotein by preparing a protein preparation from a glycoprotein in a medium for analysis and analyzing enzymatically treated samples alongside a media sample background control. The protein preparation is divided into multiple protein samples, including a sample with no enzyme, a sample treated with at least β-galactosidase, and another sample treated with at least β-galactosidase and α-sialidase.
Galactose amounts in the samples are quantified using HPAEC-PAD chromatography. The method assigns interpretive meaning to each measured galactose amount: the no-enzyme sample reflects galactose from the medium; the β-galactosidase-treated sample reflects galactose uncapped with a sialic acid residue in the protein preparation in addition to galactose from the medium; and the β-galactosidase and α-sialidase-treated sample reflects galactose capped with a sialic acid in addition to the galactose from the medium and uncapped by sialic acid.
Based on these quantified amounts, the method determines the percentage of capped galactose in the glycoprotein. The disclosure further describes extensive method development and validation, including calibration linearity, limits of quantification, repeatability, intermediate precision, accuracy recovery, robustness, sample stability, and insensitivity to digestion time.
Claims Coverage
The document provides one independent claim. It defines a multi-sample, enzyme-comparison workflow, HPAEC-PAD quantification of galactose, and determination of percent capped galactose, with an explicit linearity requirement of R2≥0.99.
Enzymatic sample partitioning for sialic-acid capping states
Dividing the protein preparation into protein samples including a sample with no enzyme, a sample treated with at least β-galactosidase, and a sample treated with at least β-galactosidase and α-sialidase, followed by incubating the plurality of protein samples.
HPAEC-PAD galactose quantification mapped to capped and uncapped states
Quantifying the galactose amount in each of the samples using HPAEC-PAD chromatography, where the samples reflect galactose from the medium, galactose uncapped with a sialic acid residue in the protein preparation in addition to galactose from the medium, and galactose capped with a sialic acid in addition to galactose from the medium and uncapped by sialic acid.
Percentage calculation of capped galactose with linearity constraint
Determining the percentage of capped galactose in the glycoprotein based on the galactose amounts in the samples, wherein the method has a linearity of R2≥0.99.
Overall, the independent claim centers on generating three enzyme-treated and untreated sample conditions that distinguish medium-derived galactose, uncapped galactose after β-galactosidase treatment, and sialic-acid-capped galactose after β-galactosidase plus α-sialidase treatment, followed by HPAEC-PAD quantification and calculation of percent capped galactose with linearity R2≥0.99.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Example application to recombinant α1-proteinase inhibitor (recAlpha-1) with use of fetuin controls for expected capping ratios.
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