Protein Analytical Services

Assay & Case Studies

• Qualitative & Quantitative Analysis

  Qualitative & Quantitative Analysis
  Applications	Assay list	Benefits
  Purity Analysis	SDS-PAGE	High acceptance
  	CE-SDS	High accuracy Automated High resolution
  	HPLC	Broad applicability
  	UPLC	Ultra-high pressure resistance Minimal delay volume Minimal sample residue
  Routine molecular weight detection	Non-deglycosylated detection: Intact mass Reduced mass	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
  Characterization of heavily glycosylated proteins	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
  Characterization of complex formation under the native state	Native mass	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
  Characterization of the complete protein sequence	Sequence coverage	Minimal impact of protein purity 100% sequence coverage
  	in-gel sequence coverage	Qualitative analysis of the band of interest
  Characterization of protein N-terminal and C-terminal sequences	N/C-terminal sequence	Confirm full protein expression Detect breaks in protein expression Identify N/C-terminal modifications

  Case 1: 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

  A

  

  B

  

  C

  

  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

  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

  Case: 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.

• Impurity Analysis

  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: 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

  
• Molecular Interaction Analysis

  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

  Case 2: 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

  
• Antibody Developability Analysis

  Antibody Developability Analysis
  Applications	Assessment Parameters	Assay List
  Functional screening	Affinity binding	BLI/SPR
  Stability monitor	Thermal stability	DSF
  Colloidal stability	SLS/DLS
  Solubility/immunogenicity prediction	Hydrophobicity	HIC-HPLC
  Aggregation/stability/solubility prediction	Self-interaction	AC-SINS
  Stability/PK/potency/immunogenicity prediction	Charge variants	icIEF/CEX-HPLC
  PK/Off-target/toxicity prediction	Polyspecificity	BVP assay
  Half-life prediction	Clearance	FcRn Binding – pH 6.0/7.4

  Why more and more ADD projects conduct early-stage developability assessment?

  Challenge

  • ~50% of Ab leads fail due to poor developability.
  • Problems often emerge at late stage, causing delays, wasted CMC cost, and lost candidates.

  Solution

  • From your sequence, Genscript delivers your antibody developability data in 8 business days, with high-precision instruments matching TOP20 Pharma standards, and competitive price 50-70% lower than other vendors.

  Results

  • Early-stage developability screening flags high-risk leads, reducing failure & cost, enabling faster informed lead prioritization.
  • In AIDD, rapid biophysical validation accelerates model iteration.

  Case: Immunogenicity prediction – mAb – Charge variants assessment by icIEF

  Charge heterogeneity is a key factor in antibody developability. In this case, icIEF revealed a dominant main peak (pI 7.527, 64.81%) with minor acidic and basic variants. The profile shows overall molecular homogeneity, while highlighting variants that may require monitoring during process development.

  Figure 1: Charge Variants Peak

  

  Table 1: Peak Summary