Application Note: Mimicking Natural Antibody Recycling for In Vitro Pharmacokinetics Studies

FcRn Affinity Chromatography Improves Functional Characterization of Monoclonal Antibodies

The success of recombinant monoclonal immunoglobulins (IgG) as therapeutic molecules hinges on several features; among them are a specific affinity for distinct antigens and a serum half-life of up to three weeks. The pharmacokinetic properties of IgG and albumin are linked to a recycling mechanism mediated by the neonatal Fc receptor (FcRn). IgGs bound to FcRn in endosomes of vascular endothelial cells are salvaged from lysosomal degradation. This interaction is strongly pH-dependent: FcRn binds IgG in the slightly acidic environment of endosomes and then releases IgG into the blood stream at physiological pH.

Efficient IgG-FcRn binding and release under natural pH conditions ensures optimal serum half-life of endogenous IgG, but also of therapeutic antibodies. Properties of the intact antibody as well as chemical and structural heterogeneities that may arise during manufacturing and storage can impact this interaction. Current methods to evaluate IgG pharmacokinetics include mouse in vivo assays and biophysical characterizations, but these are often complicated and laborious, or a poor representation of human physiology.

Roche Pharmaceuticals developed an affinity chromatography column that uses immobilized FcRn to mimic the natural antibody recycling mechanism and assess IgG pharmacokinetics. IgG is loaded on the column at pH 6.0, and then eluted by applying a pH gradient rising to 7.4 (Figure 1). In a single run instead of multiple experiments at various pH, this method characterizes the pharmacokinetics and the half-life of IgGs, both of which correlate with FcRn column retention time.1 Furthermore, IgG mixtures can be separated based on peak pattern and retention time profiles: IgGs with different Fab domains can be distinguished, oxidized IgGs can be differentiated from native, aggregates from monomers, and antibodies with mutations in the Fc part from wild-type IgGs.1

Figure 1. FcRn affinity chromatography enables functional characterization of monoclonal antibodies. Biotinylated neo­natal Fc receptor (FcRn, green) is immobilized on sepharose streptavidin beads. IgGs (blue) bind to FcRn at pH 6.0 and are eluted in a sharp and symmetric peak when a pH gradient from 6.0 to 7.4 is applied. Retention times provide information on the FcRn-IgG interaction, which is closely linked to in vivo serum half-life.

Originally developed for in-house operations at Roche Pharma, our FcRn affinity columns are now a standardized product from Roche CustomBiotech available to research teams worldwide. The FcRn affinity column is a robust tool that can complement existing characterization methods with orthogonal information for a more in-depth analysis of IgG fractions, or in some cases replace them.

Characterize Antibody Heterogeneity

Production or storage conditions can lead to heterogeneity of therapeutic antibodies in the form of aggregation or oxidation of labile methionines, which can shorten both shelf life and serum half-life. Understanding these heterogeneities can inform development of more robust antibodies with improved stability and pharmacokinetic properties. The high separation performance of the FcRn affinity column has enabled us to separate samples containing Fc-dimeric and aggregated antibodies1 as well as antibodies of various degrees of oxidation2, providing valuable information for antibody characterization and quality control.

More Information for Critical Decisions

Differences in binding kinetics of human IgGs toward mouse and human FcRn limit the value of mouse models for pharmacokinetic evaluation of human IgGs.3 Thus, IgG-FcRn interactions are generally assessed by biophysical assays, such as surface plasmon resonance (SPR). However, changes in FcRn binding measured by SPR do not always correlate well with changes in serum half-life,4 a situation attributable perhaps to the complexity of SPR assays and to inconsistent coupling of the antibody to the sensor chip which can impair biological activity.

A general disadvantage of current in vitro methods to characterize IgG pharmacokinetics is that each measurement is performed at precisely one pH value, calling for multiple experiments to investigate the complex pH-dependency of the FcRn-IgG interaction. Methods like ion-exchange chromatography that allow applying a pH gradient, separate proteins by surface charge but provide no information on pharmacokinetics.

In contrast, FcRn affinity chromatography uses a pH gradient centred on natural IgG-FcRn interactions. Resolved peaks are easily quantified by integrating the respective peak areas, and isolated fractions can be further analysed by functional characterization, re-chromatography, or mass spectrometry. In a systematic performance evaluation using various monoclonal IgG subtypes and mutants, oxidized antibodies, and in vitro glyco-engineered mAbs, the FcRn affinity column fulfilled all measured characteristics of selectivity, stability, linearity, recovery, and precision. A suitable range of 15–45 µg IgG in 50 µL sample volume was identified.4

Resolve Influence of the Fab Domain

Recent evidence suggests that not only the conserved Fc domains, but also the highly variable Fab domains of IgG influence pharmacokinetics during interaction with FcRn.5 For example, the antibodies ustekinumab (Stelara) and briakinumab (Ozespa) target the p40 subunit of IL-12 and IL-23, but differ significantly in pharmacokinetic behavior, showing half-lives of 48 h and 137 h, respectively. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) data demonstrate flexibility of Fab arms in the FcRn-IgG complex.6 Surface charges on the Fab region of briakinumab, but not ustekinumab, stabilize interactions and prevent release of the antibody at pH 7.4, thereby shortening its serum half-life. Observed pharmacokinetic differences between these two antibodies correlate with FcRn retention times, making the FcRn affinity column a powerful tool to screen variable Fab domains with respect to their impact on serum half-life.5


As an in vitro method, FcRn affinity chromatography enables predicting IgG-FcRn interactions reliably and efficiently, affording a more nuanced pre-characterization of potential recombinant antibody pharmacokinetics. Our pre-packed FcRn affinity columns bring the power of this method in a standardized, robust format that can be qualified to complement or even replace commonly applied interaction and characterization technologies.


Roche Custom BiotechCustomBiotech Roche logo

Tilman Schlothauer, Ph.D.
Department of Protein Analytics
Large Molecules Research
Roche Innovation Center
Munich, Germany



1. Schlothauer T, et al. (2013), Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. mAbs 5:576.
2. Stracke J, et al. (2014), A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies. mAbs 6:1229.
3. Grevys A, et al. (2018), A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat. Commun. 9:621.
4. Cymer F, et al. (2017), Evaluation of an FcRn affinity chromatographic method for IgG1-type antibodies and evaluation of IgG variants. Bioanalysis 9:1305.
5. Schoch A, et al. (2015), Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics. Proc. Natl. Acad. Sci. USA 112:5997.
6. Jensen PF, et al. (2017), A two-pronged binding mechanism of IgG to the neonatal Fc receptor controls complex stability and IgG serum half-life. Mol. Cell. Proteomics 16:451.

FcRn affinity columns are for use in quality control/manufacturing process only.


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