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Glycosylation

PeptideMappingIntact

Mass Analysis

Host cellProteins

IdentityPurity & ChargeHeterogeneity

Stability

AnitbodyDrug Conjugates

Trastuzumab and Rituximab: Binding Kinetics Assessment using Biacore T200

2 | External Document © 2020 Syngene International Limited

Introduction:

The neonatal Fc receptor (FcRN) is expressed in different organs where its functions can vary. Interactions between FcRN and the Fc portion of an IgG have been linked to antibody catabolism, transport and potential tissue-specific toxicities. FcRN is best known for its ability to protect antibodies from degradation, therefore improving their half-life in circulation (Figure -1).

Fc receptor binding constitutes a major consideration for in vivo pharmacokinetic analysis of antibody-based drugs, which can be beneficial for early screening of lead candidates with favorable pharmacokinetics behaviors (Figure -2).

Sorting of FcRn—IgG complexes

FcRn binds IgG inacidified endosome

IgG dissociates atphysiological pH

Acidifiedendosome

Endocyticvesicle

Recyclingendosome

FcRn

IgGSerumprotein

Blood (physiological pH)

Monocyte or endothellal cell Lysosome

Non-receptor bound proteinsare degraded in the Iysosome

Figure-1: Human IgG receptors: American Society of Haematology (2012).

IgG

Figure-2: Nature Reviews: Drug Discovery (2007).

FcγRI

γ2 α α

FcγRIIA FcγRIIB FcγRIIC FcγRIIIA FcγRIIIB FcRn

α α γ2 α

α-GPI

α

β2m

Highaffinity

HighaffinitypH<6.5

Lowaffinity

Lowaffinity

High &Low

ITIM

External Document © 2020 Syngene International Limited | 3

The surface plasmon resonance (SPR) technique serves as an important high-throughput platform to characterize interactions and binding affinities. Biacore systems monitors the molecular interactions in real time employing the principle of SPR. The surface responds to changes in the concentration of molecules as molecules bind to or detach from the surface. This yields several quantitative parameters that include the rates of association (Kon) and dissociation (Koff), as well as the binding affinity (KD) of the interaction (Figure-3). The measurement of the kinetics of critical molecular interactions between the drug, its target and other key receptors (e.g., Fc receptors in the case of antibodies), is a precise and accurate means of comparing biosimilars and innovators.

In order to determine the mechanism of receptor and the antibody interaction, we used SPR binding assays to measure the binding of Trastuzumab and Rituximab to Fc receptors. The biotinylated FcRN was immobilized on the Sensor Chip CAP by carboxymethyl dextran and complementary ssDNA to streptavidin. The assay was optimized at various parameters like assay buffer, regeneration buffer, receptor and IgG concentration. Both samples were evaluated in triplicates over a concentration range of 125 nM to 7.8 nM (8 Doses) and the data was analyzed using BIA evaluation software version 3.1. The KD values were calculated using kinetic surface bound analysis. The equilibrium KD was calculated by fitting the curve to a 1:1 reaction binding model.

Baseline(buffer)

Biotin CAPtureReagent

Biotinylatedmolecule

Time

Res

po

nse

Analyte Regenereration

Figure-3: Overview of the receptor: ligand interaction.

Sensor Chip CAP

Analyte

Biotinylated molecule

Biotin CAPture Reagent[SA+complementory oligoto the one on the surface)

Method:

Results:

Figure-4: Representing sensogram for Trastuzumab binding to FcRn.


Figure-5: Representing sensogram for Rituximab binding to FcRn.

Table-1: Representing table showing kinetics data of Trastuzumab and Rituximab binding to Fc receptor (Set-1).

Sample ka (1/Ms) kd (1/s) KD (nM) Rmax (RU) Chi²

Trastuzumab 2.68e+06 2.83e-01 105 16.8 0.058

Rituximab 2.61e+06 2.67e-01 102 19.1 0.063

Table-2: Representing table showing kinetic data of Trastuzumab and Rituximab binding to Fc receptor (Set-2).

Sample ka (1/Ms) kd (1/s) KD (nM) Rmax (RU) Chi²

Trastuzumab 2.86e+06 4.05e-01 141 7.1 0.058

Rituximab 2.01e+06 1.57e-01 78 4.6 0.042

Table 3: Representing table showing reproducibility of Trastuzumab and Rituximab binding to FcRN in two set of experiments and it was found to be between 13-15 % of coefficient of variance.

Sample Set-1 (KD) Set-2 (KD) %CV

Trastuzumab 105 141 14.63

Rituximab 102 78 13.33

A SPR-based Fc receptor binding assay was successfully developed using Trastuzumab and Rituximab standards and evaluated as a general assay platform, which is simple, quick, and ready to apply to different types of IgG molecules with minimal modification. The assay was demonstrated to be specific, precise and accurate, and it was able to detect changes in FcRn binding between different monoclonal antibodies (IgG). Due its simplicity and high-throughput capability, the assay can successfully use to support various studies.Fc receptor is a critical contributor to the half-life of therapeutic antibodies. An accurate, precise and specific in vitro FcRn binding assay is currently required to support antibody engineering, process development, comparability studies, and biological characterization of pharmaceutical molecules. The method developed here provides a platform approach that can be readily applied to various antibodies.

Conclusion:


References:

1. Neuber T, Frese K, et al. (2014) Characterization and screening of IgG binding to the neonatal Fc receptor. mAbs. 6(4):928-42.

2. Roopenian DC, Akilesh S. (2007) FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol. 7(9):715-25.


Notes:


Notes:

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