Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function.

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.

Influence of glycan modification on IgG1 biochemical and biophysical properties.

Monoclonal antibodies (mAbs) are the fastest growing class of biopharmaceuticals. The specific therapeutic tasks vary among different mAbs, which may include neutralization of soluble targets, activation of cytotoxic pathways, targeted drug delivery, and diagnostic imaging. The specific therapeutic goal defines which interactions of the antibody with its multiple physiological partners are most critical for function, and which ones are irrelevant or indeed detrimental. In this work, we explored the ability of the glycan chains to affect IgG1 interaction with two key receptor families, FcRn and γ-type Fc receptors, as well as the influence of glycan composition on the conformation and stability of the antibody molecule. Three different glycan-modified forms of IgG1 (fully deglycosylated, hypergalactosylated and hypersialylated) were produced and characterized alongside the unmodified mAb molecule. Biophysical measurements did not reveal any changes that would be indicative of alterations in the higher order structure or increased aggregation propensity for any of the three glycoforms compared to the unmodified mAb, although the CH2 domain was shown to have reduced thermal stability in the fully deglycosylated form. No significant changes were observed for the hypergalactosylated and hypersialylated forms of IgG1 with regards to binding to FcRn, FcγRIIA and FcγRIIIA, suggesting that neither half-life in circulation nor their ability to induce an immune response are likely to be affected by these modifications of the glycan chains. In contrast, no measurable binding was observed for the deglycosylated form of IgG1 with either FcγRIIA or FcγRIIIA, although this form of the antibody retained the ability to associate with FcRn. These highly specific patterns of attenuation of Fc receptor recognition can be exploited in the future for therapeutic purposes.

Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.