Protein Analytical Service

Case1 Native mass - BsAb - Detect the non-covalently linked chain that not display in conventional mass spectrometry

One of the light chains in this bispecific antibody sample is non-covalently linked to the other chains. Fig 1A shows molecular weights of 150K, 120K, 100K, 74K, and 23K. However, conventional mass spectrometry, which operates under denaturing conditions, did not detect the 150K signal, indicating that the non-covalent interactions between proteins were disrupted under these conditions (Fig 1B). In contrast, native mass spectrometry can reflect the original state of the proteins without disrupting their non-covalent interactions, successfully detecting this light chain (Fig 1C).

A

B

C

Figure 1 SDS-PAGE and Mass analysis of BsAb1

Case 2 In-gel sequence coverage - Biotinylated protein – Identification for low concentration protein

The client requested the detection of biotinylation efficiency for protein-1. However, its concentration is far below the requirements for molecular weight and solution coverage in the standard submission guidelines, 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 (Table 1).

Figure 1 SDS-PAGE of protein-1

Table 1 Peptide coverage analysis of protein-1

Case1 Glycosylation mirror comparison – Glycoprotein – Production process optimization and selection

Ab-1 and Ab-2 are samples of the same protein obtained through different production processes (with different hosts). As shown in Fig1 & Tab1, Glycan profiling analysis can be used to study the impact of different production processes on the glycosylation patterns of the protein, facilitating process optimization and selection.

Figure 1 Reduced Mass analysis of Ab-1 & Ab-2

Table 1 Glycosylation analysis of Ab-1 & Ab-2

Case 1 SPR - Human IgG – SPR has a broader affinity detection range

As shown in Fig1 & Tab1, for medium-affinity samples (mostly antigen-antibody), the detection results (KD) of BLI and SPR are almost consistent. For high-affinity samples, due to limitations in instrument stability and sensitivity, BLI may show artificially high detection results or fail to detect. For low-affinity samples (fast on and fast off, such as most small molecules and peptides), due to sensitivity limitations, BLI may fail to detect.

Human IgG-1

Human IgG-2

Human IgG-3

Figure 1 Affinity binding curves of human IgG-1, 2, 3

Table 1 Affinity binding detailed analysis report of human IgG-1, 2, 3

Case2 BLI v.s. SPR – IgG Affinity - BLI and SPR results are comparable for most Ab-antigen interactions.

Rabbit IgG

Rabbit IgG

Mouse IgG

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 ruling out protein contamination, it was suspected that this band was a host cell protein (HCP). The band was excised from the gel and subjected to HCP analysis, which revealed a high degree of similarity to phosphopyruvate hydratase (Tab 1). Further matching of the host protein sequence showed an overall sequence coverage of 90.9%, confirming that the band was the host 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