Therapeutic ADCC⁺ Biobetter Antibody Analysis

Therapeutic ADCC⁺ Biobetter Antibody Analysis

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Antibody-dependent cellular cytotoxicity (ADCC) is an important immune mechanism for antibodies. For the past years, ADCC-enhanced therapeutic antibodies have been shown to exhibit unprecedented anti-cancer effects in vivo. During the research, characterizing ADCC activity and determining its impact on clinical efficacy is critical. With the leverage of our knowledge and rich experience in antibody engineering and glycosylation control, Creative Biolabs is committed to developing next-generation ADCC-enhanced therapeutic antibodies for our customers.

Our Expertise

Invest in advanced analytical instrumentation
Perform comprehensive ADCC⁺ antibody characterization
Develop and optimize tailored characterization
Treat and deliver data in a secure, accurate and resolute manner

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End-to-End Analysis

ADCC Assays
Glycan Assays
FcγR Binding Assays
Aggregation Characterization
Physicochemical Characterization
Octet-Based Kinetic & Affinity Characterization

ADCC Assays

Our ADCC assays can use human Peripheral blood mononuclear cell (PBMC) or NK cells as effector cells and the goal is to determine the antibody activity against specified target cell lines.

Target Cell

We have knowledge working with extensive cell banks (more than 100 qualified tumor cell lines). If you have trouble finding the right cell line to use, our technical support can help you find the most ideal cell line for your ADCC analysis.

Effector Cells

PBMCs and NK cells are examples of classical immune cells involved in the ADCC effect, which both interact with the Fc portion of IgG via the Fc receptor (primarily FcγRIIIa).

  • Primary ADCC Assay—Creative Biolabs collects fresh PBMC or purified NK cells from healthy genotypes from healthy or diseased donors using standard procedures.
  • ADCC Report Analysis—ADCC analysis based on primary effector cells can be both time consuming and expensive. Also, using PBMC is reported to have intra- and inter-assay variability, which may lead to inconsistent results. Therefore, Creative Biolabs uses engineered effector cells to react with the FcγRIIIa receptor and expresses FcγRIIIa and luciferase, which allows for optimal analytical performance by ADCC reporting bioassays.

Analytical Endpoint

  • Target cell lysis in vivo and in vitro: LDH (lactate dehydrogenase) assay / ATP release / FACS screening
  • Effector cell activation: luminescent NFAT reporter gene
  • Real-time monitoring: our platform actively analyzes target cell proliferation and death rate in the presence of effector cells and test antibodies, rather than single-end measurement. This allows for a quick, simple but sensitive detection of subtle ADCC activity.

Real-time monitoring of target cells at different time points Fig.1 Real-time monitoring of target cells at different time points.

In vitro ADCC assay Fig.2 In vitro ADCC assay.

Creative Biolabs offers a range of in-depth glycan analysis and Fc receptor binding assays to complement on your ADCC functional analysis to thoroughly characterize ADCC⁺ products.

We also provide ADCP assay service, please visit: ADCP Assay Services for more details.

Glycan Analysis

Afucosylation of IgG N-glycans can influence receptor antibodies binding affinity, and thereby, acting on ADCC activity. A thorough characterization to determine the degree of antibody defucosylation is very important for ADCC⁺ therapeutics. We offer analytical methods for N-glycans in its different formats, from intact proteins, glycopeptides, released glycans, to individual monosaccharide components. Analysis characterization is done via high-resolution precision Hydrophilic interaction ultra-performance liquid chromatography (HILIC–UPLC).

Depending on the exact objective, glycosylation analysis can be performed at different levelsFig.3 Depending on the exact objective, our glycosylation analysis can be performed at different levels.

Fc Receptor Binding (FcγR) Assays

Creative Biolabs is able to perform FcγR binding assays on defucosylated Fc fusion proteins, using our Biacore's surface plasmon resonance (SPR) and Octet Biolayer Interferometry (BLI) technology platforms. The simplicity and utility of these two platforms for the determination of binding profiles of therapeutic ADCC⁺ antibodies and FcγR are aslo demonstrated during applciation.

With the help of experienced scientists, our FcγR binding assays are performed based on the following techniques:

  • Conventional ELISA
  • Flow cytometry
  • Surface Plasmon Resonance (SPR)/ Octet Biolayer Interferometry (BLI) (recommended): give real-time unlabeled full kinetic analysis, and able to report affinity constants (KD) associated with detailed or "on" rates (Ka), dissociation rates (Kd) and/or "closed" rates

Fc receptor binding assays on the Octet system Fig.4 Fc receptor binding assays on the Octet system.

Aggregation Characterization

Antibody aggregates are considered as impurities or molecular variants and may be caused by the effects of physical factors such as light, temperature, pH, water, shear force, or reaction with excipients over time in the formulation, etc. Common aggregates include charge variants, dimers, oligomers, or forced degradation variants of the desired antibody product.

Services Techniques
Antibody Purity Analysis SDS-PAGE
Antibody Polymer Analysis Size Exclusion Chromatography (SEC)
Antibody Fragment Analysis Reversed-phase Chromatography (RP)
Antibody Isoform Analysis Capillary isoelectric focusing

Characterization of low molecular weight impurities associated with products in therapeutic monoclonal antibodies by hydrophilic interaction chromatography-mass spectrometry Fig.5 Characterization of low molecular weight impurities associated with products in therapeutic monoclonal antibodies by hydrophilic interaction chromatography-mass spectrometry.

Physicochemical Characterization

Biochemical and biophysical characterization of therapeutic antibodies to fully understand their structural and functional properties is extremely important to your research. Assessing Program Quality Assessment (PQA) is the foundation for product control strategies (specifications, comparability and stability strategies). Here at Creative Biolabs, we provide biochemical and biophysical characterization of APIs and pharmaceutical products during antibody drug development.

Primary Structure Characterization

Services Techniques
Intact Mass Confirmation Native, deglycosylated, and reduced protein electrospray ionization-mass spectrometry (ESI-MS)
Amino Acid Sequence Confirmation MS/MS sequencing of peptide maps
N- and C-terminal Variants MS/MS sequencing of peptide map
N- and C-terminal Sequencing De novo antibody sequencing
Disulfide Structure Non-reduced and reduced peptide map with MS Ellman's assay (or similar)

Biophysical Characterization

Services Techniques
Secondary structure Fourier transform infrared (FTIR) spectroscopy, circular dichroism
Tertiary structure Near-UV circular dichroism (near-UV CD) spectroscopy, differential scanning calorimetry, X-ray crystallography
Thermal stability Differential scanning calorimetry (DSC)

Chemical Modification Characterization

Analysis of post-translational chemical modifications (including the categories and sites of modification) is particularly important for cell biology as well as clinical transformation. At Creative Biolabs, we offer various off-the-shelf chemical modification/ characterization services to assist your scientific research.

  • Glycosylation Analysis
  • Phosphorylation Analysis
  • Deamidation and Oxidation Analysis
  • Disulfide Bridges & Free Sulfhydryl Groups
  • Antibody C-terminal Lysine Variants Determination

Octet-Based Kinetic & Affinity Characterization

Creative Biolabs offers Octet System-based analysis services for antibody binding kinetic characterization. Octet complete kinetic analysis service allows the recording and identification of antibody drugs for their batch consistency and drug stability.

Available Octet Systems

The RTX-DE(+fu) antibody was glyco-optimized for ADCC and harbored an amino acid to exchange S239D-I332E against CD20. Enhanced FcγRIIIa binding and ADCC activity were observed.

In addition to identifying binding partners to target molecules, Creative Biolabs also provides quantitative information regarding following properties:

Specificity

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Concentration

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Kinetics

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Affinity

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Creative Biolabs is dedicated to developing the right bioassay to help you further your ADCC⁺ antibodies research. For more details about our Therapeutic ADCC⁺ Biobetter Antibody Analysis service, please do not hesitate to contact us.

RESOURCES

Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details. 

All products and services are for Research Use Only. Do Not use in humans.

CASE STUDY

ADCC Assay WT vs AfucoTM Mabs  Visit

Resource

Antibody Fc Engineering: Towards Better Therapeutics  Visit

ONLINE INQUIRY

Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.

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