Thursday, April 16, 2009

A parallel approach to SPR analysis provides more answers, faster.

By Laura Moriarty, PhD

Surface plasmon resonance (SPR) has revolutionized the study of the protein interactions required for the execution and maintenance of complex biological processes. SPR studies have helped to elucidate the roles that intracellular concentration, ionic environment, cofactors, and protein conformation play in maintaining those processes. Just as importantly, SPR has become a powerful tool for drug development, including the detailed evaluation of drug lead compounds, optimization of affinity protein purification methods, and rapid identification of highly specific monoclonal antibodies with high affinity for the analyte of interest.^{1, 2}

Label-free protein interaction data with SPR

SPR occurs when light interacts with a metal film placed at the interface between two media with different refractive indices, such as glass and water. SPR biosensors respond in real time to changes in the refractive index resulting from the binding and subsequent separation of two proteins. A ligand (antibody) is chemically bound to a gold layer on the sensor surface. As an analyte (antigen) in solution flowing over the surface binds to the immobilized antibody, the refractive index near the sensor surface increases, leading to a shift in the SPR angle. When a solution devoid of antigen replaces the antigen solution, the protein complex on the sensor surface dissociates, and the SPR angle shifts back. The shift in the SPR angle is measured in response units (RU) and recorded as a function of time in the form of a sensorgram These changes in refractive index are proportional to mass changes at the surface of the sensor chip, and the sensorgram displays the time course of binding. The association phase begins with the flow of analyte. When the analyte solution is replaced by buffer, the dissociation phase starts and the signal declines as analyte leaves the ligand surface (Figure 1).

Figure 1. Representative sensorgram from SPR analysis of an antibody-antigen interaction.

Optimized association, dissociation, and equilibrium constants are calculated by fitting response data for a range of analyte concentrations under identical reaction conditions to a computational model.

Surface plasmon resonance technology offers several advantages over traditional methods for analyzing protein interactions. Neither radiochemical nor fluorescent labels are required to provide real-time data on the affinity, specificity, and kinetics of interactions. This means that all compounds can be analyzed, and binding measurements are not limited only to those proteins that can be derivatized with label moieties. SPR is also a highly sensitive technique that requires much less sample than traditional techniques, while enabling the detection of picomolar levels of proteins and molecules as small as a few hundred daltons. Since SPR provides real-time measurements, it delivers kinetic data on both the formation and dissociation of the complex, unlike equilibrium-based methods.

Multiplexed SPR for increased speed and capabilities

Until recently, SPR experiments for the determination of kinetic rate constants have often been run sequentially and data collected from two flow cells, one serving as a reference and a second sample flow cell for the binding interaction. The reference cell did not contain any ligand and served to measure non-specific binding of the analyte to the surface of the chip. Following the immobilization of one ligand on the sample flow cell surface, a single concentration of analyte was flowed over the ligand and the corresponding response data were measured. The signal from non-specific binding of the antigen in the reference channel was subtracted from the sample flow cell signal. The surface was then regenerated (analyte removed) to prepare the ligand for the next concentration of analyte. This sequence was repeated until a full analyte concentration series was measured. Such a sequential approach necessitates that one flow cell always be used as a reference, rather than to produce additional binding interaction data. The end result is reduced sample throughput, as well as the risk of loss of ligand activity due to repeated regeneration cycles.

An alternate approach utilizes the ProteOn XPR36 multiplexed SPR device and One-shot Kinetics from Bio-Rad Laboratories.^{3, 4} This system integrates microfluidics with a novel optical design to create a 6 × 6 interaction array.

Figure 2. Workflow for rapid screening of hybridoma supernatants.

Up to six ligands can be injected through channels in the vertical direction and up to six analytes can be injected in the horizontal direction, producing up to 36 data points in a single experiment (Figure 2, page 22). This process also creates horizontal interspots that have not been exposed to the ligand or any of the reagents used to bind the ligand to the sensor chip (Figure 3). These interspots are used to measure the non-specific interaction of the analyte with the chip surface, bulk effects, and signal drift.

This interaction array system eliminates the need to dedicate a channel for referencing and allows all channels to be used to collect binding interaction data.

Multiplexing improves and expands the capabilities of traditional technology and workflow by enabling multiple quantitative protein binding experiments in parallel. Robust kinetic analysis of an analyte concentration series can be achieved in one experiment, producing a complete kinetic profile of a biomolecular interaction, without the need for regeneration, using a single sensor chip.

Figure 3. A schematic view of the array on the surface of the ProteOn sensor chip. Ligand is flowed across the surface in the vertical direction through microfluidic channels. The analyte is then flowed through microchannels in the horizontal direction, resulting in 36 interaction spots, colored yellow. Vertical interspots are colored green and horizontal interspots are pale blue.

Rapid monoclonal antibody screening and ranking

Monoclonal antibodies (mAbs) and their fragments are paving the way for rational design of a new generation of pharmaceuticals, as well as continuing their role as a mainstay in diagnostic testing methods. The production of antibodies for these applications requires the development of fast, high throughput methodologies for screening and selecting appropriate candidate antibodies for development. These candidates must have very high affinity for the target, as well as high specificity and low cross reactivity. However, traditional methods for screening potential mAb candidates are slow and expensive, usually requiring purification of the antibody from the monoclonal hybridoma supernatants before screening can occur. These methods can require time consuming and expensive preparation of labeled ligands, with the added risk that the presence of the label will alter the interaction between the antibody and its ligand.

The 6 × 6 interaction array design of the ProteOn XPR36 multiplexed SPR system lends itself ideally to very rapid screening of hybridomas for monoclonal antibody production. The process begins with the immobilization of a purified capture antibody (in this case anti-mouse IgG) in the six parallel channels (Figure 2, page 22). Crude hybridoma supernatants from six different clones raised against an antigen of interest, in this case interleukin 9 (IL-9), are then injected orthogonally. Finally, the purified antigen of interest is injected in the same orientation as the original capture antibodies, at five different concentrations, with buffer only being injected in channel six. The resulting 36 sensorgrams (arranged in sets of six representing the six different hybridoma IL-9 clones) provide a kinetic profile detailing the kinetics of interaction between the IL-9 analyte and the monoclonal antibodies contained in the six different hybridomas (Figure 4). The data can be analyzed using a variety of computational interaction models. In this case the 1:1 Langmuir binding model was used to determine association, dissociation and equilibrium constants (ka, kd and KD). The antigen-antibody pair is removed from the capture antibody using dilute phosphoric acid, and the array is then ready for another 6 × 6 analysis of a second panel of hybridoma supernatants.

In this manner, hundreds of hybridoma supernatants can be rapidly screened in consecutive cycles of analysis, using the same sensor chip. The antibodies expressed by the hybridomas can then be ranked by the affinities and dissociation constants determined from the kinetic data (Table 1, page 24). In this illustration of the technique antibodies from 20 hybridoma supernatants were captured and their kinetic constants determined. Equilibrium dissociation constants (KD) were calculated using the ratio of kd/ka, and the table shows the results for five of the selected clones that produced antibodies with similarly high affinities (KD < 10 nM). Antibody from clone 2 has the highest affinity ranking, since it had a slightly higher affinity (lower KD) and lower dissociation rate constant (kd), than the others. This ranking process enables selection of the most promising hybridomas for more detailed kinetic analysis.

Identifying clinically valuable mAbs

The multiplex, One-shot Kinetics approach of the ProteOn XPR36 system has been used to illustrate how high-affinity antibodies for a clinically relevant protein can be identified very quickly.¹ Interleukin 12 and the more recently discovered IL-23 and IL-27 constitute a unique family of cytokines which regulate cell-mediated immune responses, and they may have a central role in the development and progression of cell-mediated autoimmune diseases, including multiple sclerosis, uveitis, diabetes, and arthritis. Targeting cytokines of the IL-12 family pharmacologically could therefore be useful in the modulation of these diseases.⁵ Nahshol et al (2008) describe how they screened 192 hybridoma supernatants from mice immunized with human IL-12 in just 14 hours, including assay setup, instrument time, and data analysis time, using the ProteOn XPR36 system.¹ Several mAbs with very high affinity (KD < 0.1 nM) were identified and these have gone on to further research on specificity and epitope mapping.

Conclusion

Multiplexed SPR and One-shot Kinetics are superb tools for accelerating the analysis of protein interactions such as the screening of monoclonal antibodies, enabling the analysis of six hybridoma supernatants simultaneously. The ProteOn XPR36 system can be set up to screen almost 200 supernatants in 14 hours, in one experiment, using a single sensor chip. This approach eliminates the need to purify antibodies from their supernatants prior to analysis, and can be used to identify clinically relevant mAbs with very high affinity and specificity for the targeted antigen, at earlier stages in the development workflow.
Laura Moriarty, PhD, is a product manager at Bio-Rad Laboratories. She is responsible for driving new applications for the ProteOn product line and managing collaborations with both academia and industry.

References

1. Nahshol, O. et al. Parallel kinetic analysis and affinity determination of hundreds of monoclonal antibodies using the ProteOn XPR36. Anal. Biochem. 383 52-60 (2008).

2. Bronner, V., Nahshol, O., Bravman, T. Evaluating Candidate Lead Compounds by Rapid Analysis of Drug Interactions with Human Serum Albumin. Am. Biotechnol. Lab. 26(5) 14-15 (2008).

3. Bravman, T., et al. Exploring "one-shot" kinetics and small molecule analysis using the ProteOn XPR36 array biosensor. Anal. Biochem. 358 281–288 (2006).

4. Bronner, V., et al. Rapid optimization of immobilization and binding conditions for kinetic analysis of protein–protein interactions using the ProteOn XPR36 protein interaction array system. Bulletin 5367, Bio-Rad Laboratories, Inc. (2006).

5. Kang, B.Y., Kim, T.S. Targeting cytokines of the interleukin-12 family in autoimmunity. Curr. Med. Chem. 13 1149–1156 (2006)

E-mail: