Development and applications of AlphaScreen-based FcRn binding assay to characterize monoclonal antibodies.

IgG antibodies are important pharmaceutical molecules that successfully treat a variety of human diseases. The neonatal Fc receptor (FcRn) interacts with IgG Fc in the CH2-CH3 domain and plays a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody pharmacokinetics in IgG engineering and screening during the early optimization stage. In addition, it could be useful in biological characterization studies for antibody minor variants, process optimization, and comparability study at later stages of antibody development. Here we developed a homogeneous AlphaScreen-based FcRn assay to assess the binding of FcRn to IgG antibody in vitro. The assay is found to be accurate, precise, specific, and simple: donor beads loaded with FcRn and acceptor beads loaded with IgG1 mAb1 are mixed together with sample IgG at various dilutions and incubated for 1h before acquiring data with a fluorescence reader. This assay can run up to four samples per plate in 2h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies.

Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a relevant characteristic to measure for a number of therapeutic monoclonal antibodies (mAbs) under development. ADCC is a mechanism by which antibody-opsonized, infected, or cancerous cells are destroyed by FcγRIII (CD16)-expressing effector cells. Here we describe three methods that can be used to quantify the ADCC activity of mAbs by measuring distinct aspects of the ADCC mechanism.

Evaluation of semi-homogeneous assay formats for dual-specificity antibodies.

Dual specificity antibodies such as bispecific and Dual Action Fab (DAF) antibodies are emerging therapeutic products with powerful therapeutic potential. New bioassay formats are needed in order to monitor their dual biological activities. Here we describe the optimization and development of a "bridging" binding method in semi-homogenous (SH) assay format for a bi-specific antibody. In the SH assay format, the antigen and secondary antibodies are mixed directly with the sample solution. The bound complex is then captured onto a microtiter plate coated with the other antigen and subsequently detected by colorimetric methods. We demonstrated that an SH assay achieved comparable dynamic range, quantitation, and specificity to the conventional assay format where each reagent is added sequentially followed by separate washing and incubation steps. The SH assay requires fewer wash steps and the overall assay time is shortened by half, which translates to improved efficiency without any requirement for new equipment and reagents. Using the SH assay format, we also demonstrated that a DAF antibody which can bind to two different antigens with either arm could bind both antigens simultaneously in vitro. The SH assay format has broad application as an assay platform for assessing antigen binding properties efficiently.

Development of an ELISA based bridging assay as a surrogate measure of ADCC.

Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing. To provide an alternative to the cell lysis based ADCC assay, a non-cell based measure of ADCC activity was developed to determine the ADCC activity of an anti-CD20 mAb, which measures the ability of the mAb to bind to CD20 antigen and FcγRIIIa, simultaneously. The bridging of CD20 and FcγRIIIa is an essential interaction for the initiation of ADCC activity. This ADCC bridging method is simple, offers the ease of use of a standard ELISA, and shows reproducible dose-response curves in the concentration range of 50-1000 ng/mL. With interassay variability of 7-10% and recovery of 89-115%, the assay demonstrates acceptable precision and accuracy. The assay is able to detect degradative changes in anti-CD20 mAb samples subjected to light and acid exposure, suggesting that it is suitable for use as a stability-indicating method. The assay is also sensitive to mAb fucose levels. A linear relationship between the ADCC bridging assay and the cell lysis ADCC assay was demonstrated, strongly suggesting that the ADCC bridging assay can be used as a surrogate measure of ADCC.

Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

Accurate determination of succinimide degradation products using high fidelity trypsin digestion peptide map analysis.

We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.