Therapeutic CDC⁺ Biobetter Antibody Analysis

Therapeutic CDC⁺ Biobetter Antibody Analysis

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Fc-mediated mechanisms complement-dependent cytotoxicity (CDC) is an important pathway to research for successful therapeutic intervention. Powered by our professional scientists and their years of field experience, Creative Biolabs is able to perform highly accurate and reliable CDC analysis to study the cytotoxic effects of CDC⁺ antibodies. With our cutting-edge CDC analysis platforms, we can assess the CDC cytotoxicity of your treatment candidates in a speedy and high-throughput manner.

Our Expertise

Invest in advanced analytical instrumentation
Perform comprehensive CDC⁺ 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

C1q Binding Analysis
Cell Death Analysis
FcγR Binding Assays
Aggregation Characterization
Physicochemical Characterization
Octet-Based Kinetic & Affinity Characterization

Before CDC Analysis

Creative Biolabs has extensive experience in CDC analysis and we believe we could help you to customize the best analysis strategy with key factors identified to influence CDC activity. Important factors include:

Antibody isotypes

Certain antibody isotypes have better CDC capabilities than others. Generally, CDC ability is shown in the order of G3>IgG1 >>>IgG2≈IgG4. Also, the substitution of a specific amino acid in the hinge region or CH2 domain of an antibody can improve overall CDC activity.

Expression level of the antigen on the target cell

A desired cell line should express enough target antigens for antibody to bind and recruit complements. Complement activation cannot be initiated without the existence of an appropriate number of antigens.

Expression of complement modulators on target cells

A desired cell line can express surface proteins in a way to either inhibit or regulate complement activation. Some of these membrane binding modulators include CD46, CD55, and CD59.

Complement source

Complement is usually found in serum but can be also added to the assay to promote CDC. Therefore, it is important to know the biological source of the serum to ensure it is compatible with the target cells. We provide a complete panel of complement components that depends on the customizable selection of the analyte, such as C2, C4b, C5, C5a, C9, factor D, MBL and factor I. Please visit: Complement Component Assay Services for more details.

Afuco™ technology platform Fig.1 Real-time monitoring of target cells at different time points.

Afuco™ technology platform Fig.2 In vitro ADCC assay.

End-to-end CDC Analysis

Our experts can help you understand the functional characterization requirements of effectors, define successful CDC characterization strategies for biobetters, and perform the experiments. Our CDC analysis services include:

C1q Binding Analysis

CDC is an effector function of IgG and IgM antibodies. When these antibodies bind to surface antigens, the CDC is triggered, resulting in the formation of membrane attack complex (MAC) and target cell lysis. In the process of the complement cascade, one complement system component, C1q, is involved in binding to the antibody Fc. In other way, C1q binding assays activate the C1 complex by binding to multiple IgG molecules complexed with an antigen.

Creative Biolabs offers various C1q binding assays to support the production, characterization, process development of studies on CDC⁺ mAbs. Currently, our team has already successfully utilized the sensitive Surface Plasmon Resonance (SPR) technology for C1q binding assays.

SPR analysis of C1q bindingFig.1 SPR analysis of C1q binding.

Quality-controlled complement sources

At Creative Biolabs, we have refined the protocol for quality control of complement sources, per the descriptions on ICH Guide Q6B. Common sources of complements include humans, non-rodent animals, and/or rodents that used for characterization or therapeutic candidate screening purposes. In addition, with our extensive understanding of the CDC studies, Creative Biolabs can help you design the right experiment to determine the CDC potential of your therapeutic antibody.

Creative Biolabs also provides a complete solution for complement function analysis. Please visit: Complement Function Assay Services for more details.

Cell Death Analysis

CDC⁺ therapeutic antibodies exert a cytotoxic effect through a complement-mediated mechanism. Thus, a useful attribute of characterizing CDC antibodies is to assess whether they have the ability to kill cells with which they bind. Creative Biolabs has developed a high-throughput flow cytometry system for cell death assays that can perform extensive multi-dose characterization of a range of drug candidates in a short period of time.

Dose-response curves and EC50 values of CDC induced by the CD20 mAbFig.2 Dose-response curves and EC50 values of CDC induced by the CD20 mAb.

Fc Receptor Binding (FcγR) Assays

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

With the help of experienced scientists, Creative Biolabs introduces a novel, reliable and highly flexible platform for evaluating FcγR binding properties. Our FcγR binding assays is 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 systemFig.3 Fc receptor binding assays on the Octet system.

Aggregation Characterization

Antibody aggregation is considered as impurities or molecular variants and, potentially, 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 spectrometryFig.4 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 ultimate 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 primarily 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) of antibodies is particularly important for cell biology as well as clinical transformation. At Creative Biolabs, we offer a variety of 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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Our goal is dedicated to helping our clients get meaningful results through powerful and consistent approaches. Our data provide functional characterization of the biological activity of your CDC⁺ antibodies and direct preclinical transitions from in vitro studies to in vivo studies.

For more details about our Therapeutic CDC⁺ 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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