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IgG Structure and possible biological activity

Take a mAb as shown below for example, a thorough package of bioactivity characterization should be tailor-made for each product.

Antibodies Characterization Services

Chiu M L, Goulet D R, Teplyakov A, et al. Antibody structure and function: the basis for engineering therapeutics[J]. Antibodies, 2019, 8(4): 55.

Binding Kinetics (Fab region and Fc region)

Antibody-antigen (Ab-Ag) interaction is critical for the function of antibody-related therapeutics. According to FDA guidelines for bispecific antibody development, the affinity, on- and off-rates for both targets should be characterized.

The FcγR subclass is involved in interactions with Fc-containing therapeutics. A comprehensive characterization for Fc–FcγR is indispensable for any Fc-involving biologics. FcRn is involved in the turnover of Fc-related biologics, while C1q affinity is related with CDC function, both of which should be included in the Fc characterization package.

The commonly used methods for affinity characterization are surface plasmon resonance (SPR), biolayer interferometry (BLI) and enzyme-linked immunosorbent assays (ELISA). The former two are label-free tools, providing real-time association and dissociation rates. SPR is previously dominant, while BLI is recently recognized in the USP and more and more prevalent in industries given its high throughput and friendly maintenance. Notably, BLI could do a bit more, i.e. C1q affinity, than SPR. The end-point ELISA is more cost-effective but somewhat limited and lengthy. A complete package of Fc characterization by BLI wins more and more favor in biologics development.

Effector Function

  • The common Fc effector functions are antibody- dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP). For some biologics, these effector functions are weapons against diseases, while for some they might be adverse effects. Thus, ADCC, CDC and ADCP should be included in the characterization package as appropriate.

Other Functional/Immunological Activity

Biologics are a vast mine for developing weapons against diseases. There are various complicated mechanisms of action (MOA), involving anti-proliferation, receptor internalization, ligand blockade, apoptosis, neutralization, enzyme activity, etc. Recent years have witnessed the rise and ongoing digging for immunomodulatory drugs, especially immune checkpoint agents, such as anti-PD-1, anti-CTLA4, anti-TIM3, etc. Mixed lymphocyte reaction (MLR) is a recognized and necessary assay for evaluation of the T cell-activating capacity of immunomodulators. Our services are summarized in the table below. We are looking forward to helping you out in the bioactivity characterization(cat no.SC2071).

Characterization items Services Method
Binding Kinetics/Affinity FcγRs affinity BLI; ELISA; SPR
FcRn affnity BLI; HTRF; SPR
C1q affnity BLI; ELISA
Ag-Ab affinity;
Kon/Koff (kinetics)
ELISA,BLI
customized assay NA
Effector Function ADCC PBMC-based;
NK-based;
Reporter-based
CDC Complement-based
ADCP Reporter-based;
Macrophage-based
TDCC CD8+ T-based
Apoptosis FACS
Other Functional/ Immunological Activity MLR cytokine release (ELISA/HTRF)
cytokine release ELISA/HTRF
IgG4 Fab arm exchange FRET/LC-MS
Cytokine storm MSD; Luminex
anti-proliferation cell-based assay
Enzymatic assay Endpoint Enzyme Activity Assay
blockade/neutralization cell-based assay
Internalization FACS
customized assay NA
Head-to head study customized assay case by case

References:

[1] Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, ICH, 1999, August

[2] Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use, FDA, 1997, February

[3] Guideline on development, production, characterization and specification for monoclonal antibodies and related products, EMA, 2016, September

[4] Chiu M L, Goulet D R, Teplyakov A, et al. Antibody structure and function: the basis for engineering therapeutics[J]. Antibodies, 2019, 8(4): 55.

[5] Guidence for Industry: Bispecific Antibody Development Programs, FDA, 2019, April

[6] USP <1108>Assays to Evaluate Fragment Crystallizable (Fc)-mediated Effector function

[7] Zahavi, David, and Louis Weiner. "Monoclonal antibodies in cancer therapy." Antibodies 9.3 (2020): 34.