Cell Surface Glycan Profiling Using PNGase F and Mass Spectrometry
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Solution Overview
Problem
There is a need for improved methods to measure and identify glycans, particularly N-glycans, expressed on the surface of cells, as existing methods are inadequate for assessing cell surface glycosylation patterns.
Innovation Solution
Methods involving the incubation of a cell composition with PNGase F to release glycans, followed by labeling and analysis using mass spectrometry to determine the presence, absence, identity, and level of glycans, while maintaining cell integrity and minimizing cell lysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are homogenized or sonicated prior to incubation, then cell lysis is enhanced and glycan release is improved, but cell integrity is compromised and glycan profiles become contaminated with intracellular glycans
Solution Approach 1:
The patent extracts only surface glycans from intact cells using PNGase F treatment, separating surface glycans from intracellular glycans. This extraction approach allows measurement of surface glycan profiles without compromising cell integrity or introducing contamination from cell lysis products.
Solution Approach 2:
PNGase F serves as an intermediary enzyme that specifically targets and removes N-glycans from the cell surface without lysing the cells. This intermediary agent enables glycan release while maintaining cell membrane integrity, allowing subsequent analysis of surface glycan profiles.
2Measurement precision
If cells are incubated with proteases to release glycans, then glycan release is enhanced, but cell surface proteins are degraded and glycan profiles are contaminated
Solution Approach 1:
The patent extracts only surface glycans from intact cells using PNGase F treatment, separating surface glycans from intracellular glycans. This extraction approach allows measurement of surface glycan profiles without compromising cell integrity or introducing contamination from cell lysis products.
Solution Approach 2:
PNGase F serves as an intermediary enzyme that specifically targets and removes N-glycans from the cell surface without lysing the cells. This intermediary agent enables glycan release while maintaining cell membrane integrity, allowing subsequent analysis of surface glycan profiles.
3Measurement precision
If cells are incubated with detergent agents to extract membrane proteins, then glycan release is improved, but cell membrane integrity is compromised and glycan profiles are contaminated
Solution Approach 1:
The patent extracts only surface glycans from intact cells using PNGase F treatment, separating surface glycans from intracellular glycans. This extraction approach allows measurement of surface glycan profiles without compromising cell integrity or introducing contamination from cell lysis products.
Solution Approach 2:
PNGase F serves as an intermediary enzyme that specifically targets and removes N-glycans from the cell surface without lysing the cells. This intermediary agent enables glycan release while maintaining cell membrane integrity, allowing subsequent analysis of surface glycan profiles.
4Difficulty of detecting and measuring
If conventional glycan analysis methods are used, then measurement capability is provided, but sensitivity and specificity for identifying glycan species are insufficient
Solution Approach 1:
The patent replaces conventional mechanical or immunological detection methods with mass spectrometry-based analysis. This substitution enables high-resolution identification and quantification of glycan species, providing superior sensitivity and specificity for glycan profile assessment.
Solution Approach 2:
The patent changes the detection parameter from conventional methods to mass spectrometry, enabling precise measurement of glycan mass-to-charge ratios. This parameter change allows accurate identification of glycan structures and their relative abundances on cell surfaces.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These methods enable precise assessment of cell surface glycan profiles with high sensitivity and specificity, allowing for the identification of various glycan species and their levels, which is crucial for understanding cellular functions and manufacturing consistent cell compositions.
Implementation Method 1
incubating a test composition comprising a plurality of cells under conditions to release one or more glycans from the surface of cells
Implementation Method 2
release one or more glycans from the surface of cells
Implementation Method 3
determining the presence, absence, identity and/or level of glycans present in the sample
Data Source
AI summary
Provided herein are methods for assessing cell surface glycans, e.g., N-glycans, by assessing a sample of released surface glycans, and determining the presence, absence, or level of glycans present in the sample. Also provided are methods of assaying and/or evaluating a cell composition by assessing the cell surface glycan profile of the cell composition and comparing the profile to a reference sample. Methods for manufacturing and/or culturing a plurality of cell compositions having consistent surface glycan expression with low variability are also provided.