Biocytogen has established a service workflow for high quality characterization analysis of antibody drugs based on a platform fitted with comprehensive and advanced testing instruments, and 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, and have accumulated rich experience in biophysical characterization of protein in order to support antibody drug development. Furthermore, our team can accomplish method transfer, optimization and validation rapidly upon specific customer demand.
Examples of some analytical methods:
|Type of Characterization Analysis||Test Item||Test Method|
|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|
|Glycan profiling and content||LC-MS|
|Nonglycosylated heavy chains, small molecular fragments||rCE-SDS|
|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 by 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
Affinity is one of the important parameters for evaluation of intermolecular interactions, in particular, in the antibody drug screening stage; affinity is an important measure of understanding molecular recognition and bioprocesses and 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, etc.
(2) Molecular Interaction Analysis. The antibody is a finished product and representative data of the affinity measurement to human CTLA4(A) or Cynomolgus Monkey CTLA4 (B) proteins were determined using the SPR technology.
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 an important prerequisite of determining if a new antibody can be further studied in the early antibody drug development, and such analysis is a key step of determining if the candidate molecule can be made into a drug.
Biocytogen’s characterization analysis service team is equipped with Agilent 1260 Infinity liquid chromatograph system, Maurice one -stop cIEF and CE-SDS dual function analysis system and WATERS XEVO G2-XS UPLC-QTOF, and has established a well-appointed physicochemical analysis platform for characterization of physicochemical properties of an antibody in development stage and for guidance on next development process of the antibody drug.
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 in case poor test results arise.
Endotoxin assay is to determine 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.