Antibody structure and half-life: fast and reliable insights with FcRn Affinity Columns

Half-life is an essential pharmacokinetic attribute of therapeutic antibodies and thus, tools to accurately predict their clearance can advance development of novel therapies. An important determinant of the serum half-life of immunoglobulins is binding to neonatal Fc receptor (FcRn). In vivo, FcRn binds IgG at an acidic pH (<6.5) and then releases it at neutral or physiological pH to unidirectionally ferry it across epithelial cells from the lumen-facing surface to interstitial space or the bloodstream while avoiding lysosomal degradation.¹ A simple and robust device to characterize FcRn binding to antibodies that overcomes limitations of technologies like surface plasmon resonance (SPR) immunoassays constitutes a versatile method to standardize and speed up half-life analyses. 

SPR immunoassay format limits resolution of antibody complexity and heterogeneity

The interaction of an antibody with FcRn is complex. Binding takes place at the Fc region of both antibody heavy chains, plus FcRn can dimerize, giving rise to complexes with 4 FcRn molecules binding 3 antibodies or any permutation of that construct (Figure 1). Given their planar nature, SPR immunoassays simply cannot capture the three-dimensionality of this interaction. Any adjustments to the format of the immunoassay – immobilizing the antibody instead of FcRn or increasing the density of the immobilized reagent – cannot compensate for this spatial limitation and thus, SPR assays cannot reflect the full heterogeneity of the FcRn-mAb interaction.

Furthermore, antibodies with the same Fc region but different complementarity determining regions generate very different binding signals even when measured on a highly standardized SPR assay setup. The outcome is an apparent mismatch between pharmacokinetic half-life and FcRn affinity. Close examination with a hydrogen-deuterium exchange assay, however, reveals that the Fab region of the antibody also interacts with the FcRn bound to the Fc region and failure of the SPR assay to detect that interaction complexity leads to the observed mismatch.

FcRn Affinity Columns simplify antibody characterization

One way to address this mismatch and obtain a full characterization of antibodies would be to employ an array of analytical setups for each therapeutic construct developed, but this would be time-consuming and labor-intensive. Instead, an affinity column with FcRn in the solid phase could combine into a single assay the immobilization and binding architecture of SPR with the functional segregation capacity of chromatography. Such a column is now available from Roche CustomBiotech. Biotinylated recombinant human FcRn are immobilized to streptavidin Sepharose beads in the column. Leveraging the same pH-dependent binding and release behavior of the antibody-FcRn interaction observed in vivo, the immobilized FcRn binds antibodies at the top of a column at pH 6.0. Then, an elution gradient to pH 7.⁴ is applied to release the antibody at the bottom of the column. The result is a sharp, symmetric, and pH-dependent elution profile for the antibody.

FcRn Affinity Columns streamline therapeutic antibody development

Structure-function correlations revealed by FcRn affinity chromatography help predict potential behavior of antibodies in vivo and thus, facilitate their selection for ongoing development. The following are a few examples of these insights.

The Fab portion of an antibody causes a charge-dependent late retention on FcRn Affinity Columns that correlates with a shorter half-life

The Fab region of an antibody contributes to FcRn binding. Seven different IgG1 antibodies applied natively to a FcRn Affinity Column show unique retention times. However, after enzymatically cleaving the antibodies into their Fab and Fc parts, the Fab portions elute without binding to the column solid phase and all Fc portions have very similar retention times.² The influence of the Fab region creates a charge-dependent delay in retention time which correlates with a shorter half-life. Ustekinumab and Briakinumab are antibodies that target the same protein in humans but differ significantly in clearance. Briakinumab with a greater binding site charge elutes at a higher pH (longer retention time) and has a half-life of just 8 days compared to the 22 days of Ustekinumab. Engineered hybrids of the two antibodies exhibit intermediate retention and pharmacokinetic half-time such that together, the 4 antibodies align along an inverse correlation between retention time and half-life (Figure 2).³

A correlation between posttranslational modifications of antibodies and retention time on FcRn Affinity Columns can be used to screen batches

Posttranslational modifications like methionine oxidation, aggregates, and glycovariances can lead to heterogenous antibody batches and impact in vivo behavior. That heterogeneity can be deconvoluted and characterized as a correlation to retention time on a FcRn Affinity Column, transforming the column into an efficient screening tool. Retention time data are used to identify and validate a non-critical retention time window to discriminate and remove antibody samples that fall outside of the window boundaries and thus, are likely to include posttranslational modifications that significantly impact antibody pharmacokinetics.⁴

Retention time on a FcRn Affinity Column helps anticipate the direction of a shift in half-life for an engineered antibody

The FcRn Affinity Column can also be used to investigate antibody engineering. The engineered antibody mAb7-AAA is used as a standardized control in the literature because it silences the FcRn-antibody interaction. Thus, it shows an elution peak with no retention time. The wildtype antibody shows a retention time of roughly 40 minutes, while the engineered antibody mAb7-YTE has a shifted retention of almost 60 minutes (figure 3, A).² These differences correlate with antibody half-life, such that the FcRn Affinity Column can be used to approximate the half-life of engineered antibodies. Importantly, the column also allows identifying engineered antibodies with binding affinities that shift their elution beyond physiologically relevant pH, which worsens half-life in vivo. In this way, characteristic elution points on the FcRn Affinity Column for different antibodies can guide the identification of engineered antibodies that fall in the “sweet spot” of pH-dependent binding – physiologically relevant and with the desired half-life (figure 3, B).

A valuable preparative and analytical tool

FcRn Affinity Columns combine the well-known immobilization and binding setup of SPR assays with the functional segregation capacity of chromatography. Leveraging the best of both assay formats, the columns record the correlation of antibody structural variation with column retention time to open new analytical possibilities in characterizing a therapeutic antibody. Whether assessing the physiological relevance of Fab-related charge variation, oxidation, glycosylation, aggregation, and other modifications of antibodies; building an automated screening process for antibody batch quality control; or designing highly effective engineered antibodies, FcRn Affinity Columns enhance antibody half-life analysis for a more targeted and efficient development process.