| Qualitative & Quantitative Analysis
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| Applications
|
Assay list
|
Benefits
|
| Purity Analysis
|
SDS-PAGE
|
|
| CE-SDS
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- High accuracy
- Automated
- High resolution
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| HPLC
|
|
| UPLC
|
- Ultra-high pressure resistance
- Minimal delay volume
- Minimal sample residue
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| Routine molecular weight detection
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Non-deglycosylated detection:
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- Suitable for characterization of glycosylation modifications and
protein assembly
|
|
N-glycan removal detection:
- De-glycosylated mass
- Reduced and de-glycosylated mass
|
- High intensity mass spectrometry
- More accurate determination of protein chain
- Suitable for bispecific antibody identification
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| Characterization of heavily glycosylated proteins
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Denatured N-glycan removal detection:
- De-glycosylated mass(denatured)
- Reduced and de-glycosylated mass(denatured)
|
- Suitable for the identification of complete molecular weight measurement
of proteins with complex modifications.
|
|
Denatured N-glycan and O-glycan removal detection:
- De-O-glycosylated mass
- Reduced and de-O-glycosylated mass
|
- Simultaneous removal of N-glycans and O-glycans
- Elimination of their interference with protein detection
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| Characterization of complex formation under the native state
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|
- Mild detection conditions and native state preservation
- Suitable for analysis of protein complexes linked by non-covalent bonds
- Suitable for analyzing the degree of small protein aggregation
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| Characterization of the complete protein sequence
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- Minimal impact of protein purity
- 100% sequence coverage
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|
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- Qualitative analysis of the band of interest
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| Characterization of protein N-terminal and C-terminal sequences
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- Confirm full protein expression
- Detect breaks in protein expression
- Identify N/C-terminal modifications
|
Case1 Native mass - BsAb – Identification of the non-covalently linked chain
Native mass spectrometry is often used to identify the components of bispecific antibodies. The molecular weights of the marker bands in Fig 1A are 150 kDa, 120 kDa, 100 kDa, 74 kDa, and 23 kDa. Under denaturing conditions, the 150 kDa light chain was not identified by conventional mass spectrometry. This indicates that non-covalent interactions between proteins were disrupted under these conditions (Fig 1B). In contrast, native mass spectrometry reflected the original state of the proteins and successfully identified the light chain (Fig 1C).
Figure 1 SDS-PAGE and Mass analysis of BsAb1
Case 2 In-gel sequence coverage - Biotinylated protein –
Identification for
low concentration protein
Detection of biotinylation efficiency for protein-1 was requested by the customer. However, its concentration is far below the requirement and it is difficult to concentrate. Nevertheless, the SDS-PAGE bands were relatively clear (Fig 1). By excising the target band from the gel and performing coverage analysis, the biotinylation efficiency was successfully identified (Fig 2, Tab 1).
Figure 1 SDS-PAGE of protein-1
Figure 2 Fragment coverage map of protein-1
Table 1 Peptide coverage analysis of protein-1
| Site |
Mod |
M/Z |
Charge St. |
Mono Mass Exp. |
Mono Mass Theo. |
△
ppm
|
Biotinylation ratio % |
| K1296 |
Biotinylation |
980.803 |
3 |
2938. 385 |
2938.392 |
-2.36 |
99.3 |
| Protein Modification Analysis
|
| Assay List |
Benefits |
| Glycosylation mirror comparison |
- Suitable for the analysis and determination of glycosylation
sites and their respective proportions in proteins.
|
Biotinylation ratio
(peptide mapping)
|
- Targeted enzymatic cleavage for Avi tag analysis
- High accuracy
|
| Disulfide bond analysis
|
- Suitable for determination of sites and proportions of disulfide
bond
formation between cysteine residues in proteins
|
| N-glycosylation site |
- Suitable for determination of sites and proportions of N-glycosylation
|
|
PTM-phosphorylation
|
- Suitable for determination of sites and proportions of protein
modifications
|
|
PTM-deamidaton
|
|
PTM-oxidation
|
Case1 Glycosylation mirror comparison – Glycoprotein –
Production process
optimization and selection
Ab-1 and Ab-2 are the same protein produced by different hosts with different glycosylation patterns. Glycosylation mapping analysis identifies the glycosylation sites and proportions of Ab-1 and Ab-2, facilitating process optimization and selection.
Figure 1 Reduced Mass analysis of Ab-1 & Ab-2
Table 1 Glycosylation analysis of Ab-1 & Ab-2
| NO. |
Mass (Da) |
Glycan Name |
Ratio% |
| Ab-1 |
Ab-2 |
| 1 |
50063 |
N Glycosylation Man5 |
3.3% |
1.4% |
| 2 |
50086 |
N Glycosylation G0F-GlcNac |
5.9% |
4.2% |
| 3 |
50290 |
N Glycosylation G0F |
84.5% |
85.9% |
| 4 |
50452 |
N Glycosylation G1F |
6.3% |
8.6% |
Note：Mass value is from Ab-1.
| Molecular Interaction Analysis
|
| Applications |
Assay List |
Benifits |
Recommendation |
- Antigen-antibody affinity analysis
- Protein-protein interaction
- Protein-DNA interaction
- Protein-small molecule interaction
|
Bio-Layer Interferometry >>
|
- Target-free, real-time detection, high throughput
- Relatively low cost
- High sample compatibility
- Sample recovery available
|
- Suitable for the detection of supernatants
- BLI and SPR perform comparably when used to detect samples with moderate affinity(KD µM~nM).
|
| Surface Plasmon Resonance >> |
- Target-free, real-time detection, high throughput
- High sensitivity, broader affinity detection range
- Capable of detecting small molecules (100 Da)
|
- Suitable for detecting low-affinity(KD ~µM) and higher affinity (KD ~0.1 nM) samples
|
Case 1 SPR – IgG Affinity –SPR has high sensitivity and broad affinity detection range (KD = 10-3-10-15)
As shown in Fig 1 and Tab 1, SPR has higher sensitivity and a wider affinity detection range than BLI. SPR is suitable for both low-affinity (KD ~µM) and high-affinity (KD ~0.1 nM) samples, which may not be successfully detected by BLI.
Figure 1 Affinity binding curves of human IgG-1, 2, 3
Human IgG-1
Human IgG-2
Human IgG-3
Table 1 Affinity binding detailed analysis report of
human
IgG-1, 2, 3
| Ab |
Chip |
Affinity |
Test item |
Chi² (RU²) |
Ka (1/Ms) |
kdis (1/s) |
KD (M) |
| Human IgG-1 |
ProA |
Medium |
Affinity |
4.90e-02 |
1.39e+05
|
2.07e-03
|
1.49e-08
|
| Human IgG-2 |
ProA |
High |
Affinity |
3.41e-01 |
2.75e+04
|
3.59e-05 |
1.30e-09 |
| Human IgG-3 |
ProA |
Low |
Kinetics |
4.75 |
- |
- |
3.818E-07 |
Case2 BLI v.s. SPR – IgG Affinity - BLI and SPR results
are
comparable for
most antibody-antigen interactions
Most antigen-antibody interactions fall into the category of medium affinity samples, for which the detection results (KD) of BLI and SPR are nearly identical.
Rabbit IgG
Rabbit IgG
Mouse IgG
| Impurity Analysis
|
| Applications |
Test Items |
Benefits |
| Host cell protein analysis |
ELISA or LCMS |
- Provides reference for upstream manufacturing and downstream applications
|
| Residual DNA |
qPCR based |
/ |
| Residual Protein A |
ELISA |
/ |
Case 1 Host cell protein analysis – Impurity protein –
SDS-PAGE unknown band
Identification
An unknown band was detected in several production batches of the protein (Fig 1). After excluding protein contamination, this band was suspected to be a host cell protein (HCP). HCP analysis preliminarily identified the band as phosphopyruvate hydratase (Tab 1). Further sequence analysis revealed a sequence coverage of 90.9% for this protein, confirming the band as the host cell protein phosphopyruvate hydratase (Fig 2).
Figure 1 SDS-PAGE of expression sample
Table 1 Sequence coverage map report of the unkown band
Figure 2 Sequence coverage map of the unkown band
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