Characterization Analysis

Biocytogen offers antibody characterization services that allow high quality characterization analysis of antibody drugs based on intelligent use of comprehensive and advanced testing instruments. Our platform is able to provide all-around high quality one-stop testing services, ranging from early antibody screening, lead molecule confirmation to supporting IND application. In the past 2 years, we have developed various methods for bioanalysis of more than 20 target sites including bioactivity and affinity analyses, accumulating rich experience in biophysical characterization of protein to support antibody drug development. Furthermore, our team can accomplish method transfer, optimization and validation rapidly upon specific customer demand.

Examples of some antibody characterization services:

Type of Characterization Analysis Test Item Test Method

Binding Activity

Protein Binding/Inhibition ELISA (EC50, IC50)
Cell Binding/Inhibition FACS (EC50, IC50)
Molecular Interaction Analysis Affinity Biacore
Fc receptor and complement C1q binding affinity Biacore
Primary Structure Intact molecular mass or post-reduction/post-deglycosylation molecular mass LC-MS
N & C-terminal sequencing LC-MS
Disulfide bond analysis LC-MS
Sequence coverage LC-MS
PTM identification LC-MS
Glycosylation Glycosylation site LC-MS
Glycan profiling and content LC-MS
Purity Monomer/polymer purity SEC-HPLC,SDS-PAGE,WB
Nonglycosylated heavy chains, small molecular fragments rCE-SDS
Endotoxins Kit
Charge Heterogeneity Distribution of charge variants IEX-HPLC
PI and distribution of PI constituents CE-IEF/cIEF
Stability Determination of denaturation temperature Tm qPCR

(1) Binding Activity

In the early antibody drug screening, ELISA and FACS methods help us lock the candidate molecule from a great number of molecules rapidly. Furthermore, verification of binding activity at the protein level and cellular level using ELISA and FACS techniques enables an antibody drug project to proceed quickly to in vivo screening stage. Thereby Biocytogen’s characterization analysis team establishes a comprehensive biochemical analysis platform.

 

Affinity test of the anti-CTLA4 antibodies

Figure 1. Molecular Interaction Analysis of anti-CTLA4 antibodies to human CTLA. (A) or Cynomolgus Monkey CTLA4 (B) proteins were determined using the SPR technology.

Affinity is one of the important parameters for evaluation of intermolecular interactions particularly in the antibody drug screening stage. As a measure of understanding molecular recognition and bioprocesses, affinity characterization is of particular importance to pharmacodynamic evaluation. Therefore, it is essential to have a comprehensive study of intermolecular interactions, including rate and strength of intermolecular binding, when providing antibody characterization services.

Based on Surface Plasmon Resonance (SPR) technique for real-time measurement of molecule interaction without labeling, Biacore is a generally accepted gold standard for molecular interaction assays. Compared with conventional testing approach, SPR method has prominent advantages such as eliminating the need of sample marking, real-time monitoring, and high sensitivity.

Biocytogen’s characterization analysis service team is equipped with two intermolecular interaction analyzers, Biacore T200 and Biacore 8K, both of which have different features and are complementary to each other, offering an all-around service system for fast and accurate assay of intermolecular interactions.

(2) Physicochemical Analysis

Analysis of physicochemical properties of antibody drugs has become an important part of quality control in pharmaceutical industry. As one of the primary characteristic parameters of protein samples, physicochemical properties are a prerequisite of determining if a new antibody should be further studied in the early stages of antibody drug development.

Biocytogen’s antibody characterization services conduct analysis with the Agilent 1260 Infinity liquid chromatograph system, Maurice one -stop cIEF, and CE-SDS dual function analysis system and WATERS XEVO G2-XS UPLC-QTOF.

Biochemical and Biophysical Characterization Analysis Capabilities:

  • Molecular Mass Analysis
  • Intact molecular mass
  • Reduced molecular mass
  • Deglycosylated molecular mass
  • Peptide mapping
  • Amino acid sequence coverage analysis
  • Disulfide bond analysis
  • Stability study
  • Fast N-linked glycan analysis
  • N-glycosylation sites
  • O-glycosylation sites, etc.

(3)  Endotoxin Assay

Endotoxins are lipopolysaccharides (LPS) composing the cell wall of Gram-negative bacteria. As a kind of pyrogen, endotoxins can lead to inflammation and fever reactions in organisms. Therefore, in general, when involved in cell or animal testing, endotoxins have to be controlled or removed to prevent poor test results. The endotoxin assay determines the content of endotoxins by agglutination reaction between limulus blood and endotoxins. Specifically, absolutely quantitative chromogenic LAL assay is used and its detection range is 0.1–1 EU/mL.

(4) Purity Assay

▶HPLC-SEC: One kind of liquid chromatography for separation with porous gel stationary phase depending on the difference in molecular size. HPLC-SEC has better separation performance within a great range of molecular mass in a very short time, and is easy and fast to operate, resulting in reliable data and good reproducibility.
▶SDS-PAGE is a routine lab method of protein expression analysis by polyacrylamide gel electrophoresis. Proteins in a sample under test are separated in the electrophoresis gel depending on molecular mass. It is used to determine protein expression and protein purity. SDS-PAGE has a very high electrophoresis resolution, depending on sample preparation method, SDS-PAGE is classified into reducing SDS electrophoresis and non-reducing SDS electrophoresis.

(5)  Reporter Gene Assay System

Method development for biological function or bioactivity assay is one of the challenges during biotherapy R&D, for which we have established a reporter gene-based reporter cell system that can be used in high-throughput screening for any antibody drug having specific activating or inhibitory function.

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