Article pubs.acs.org/molecularpharmaceutics
Rapid Analysis of Antibody Self-Association in Complex Mixtures Using Immunogold Conjugates Shantanu V. Sule,† Craig D. Dickinson,‡ Jirong Lu,§ Chi-Kin Chow,§ and Peter M. Tessier*,† †
Center for Biotechnology & Interdisciplinary Studies, Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States ‡ Lilly Research Laboratories, Lilly Biotechnology Center, San Diego, California 92121, United States § Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46225, United States S Supporting Information *
ABSTRACT: A key challenge in developing therapeutic antibodies is their highly variable propensities to self-associate at high antibody concentrations (>50 mg/mL) required for subcutaneous delivery. Identification of monoclonal antibodies (mAbs) in the initial discovery process that not only have high binding affinity but also have high solubility and low viscosity would simplify the development of safe and effective antibody therapeutics. Unfortunately, the low purities, small quantities and large numbers of antibody candidates during the early discovery process are incompatible with current methods of measuring antibody self-association. We report a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) capable of identifying mAbs with low self-association propensity that is robust even at low mAb concentrations (5−50 μg/ mL) and in the presence of cell culture media. Gold nanoparticles are coated with polyclonal antibodies specific for human antibodies, and then human mAbs are captured from dilute antibody solutions. We find that the wavelength of maximum absorbance (plasmon wavelength) of antibody−gold conjugateswhich red-shifts as the distance between particles is reduced due to attractive mAb self-interactionsis well correlated with light scattering measurements conducted at several orders of magnitude higher antibody concentrations. The generality of AC-SINS makes it well suited for use in diverse settings ranging from antibody discovery to formulation development. KEYWORDS: monoclonal antibody, solubility, aggregation, viscosity, high-throughput, nanoparticle
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INTRODUCTION Monoclonal antibodies (mAbs) are popular drug candidates for treating a variety of diseases ranging from asthma to cancer.1,2 The primary advantages of mAbs over small-molecule compounds are their high specificity and affinity toward biological targets,3,4 as well as the relative ease of identifying antibodies via well-established in vivo (immunization) and in vitro (phage display) methods.4−13 Despite the attractiveness of mAbs as therapeutic candidates, they are generally not orally active and must be administered as liquid formulations via intravenous or subcutaneous delivery.14−16 In the case of subcutaneous delivery, mAbs must be formulated at extremely high concentrations that result in complex solution behavior including phase separation, abnormally high viscosity, opalescence, and visible and subvisible particle formation.14,17−30 Antibody particles and other types of aggregates are particularly concerning due to their immunogenicity.31,32 Therefore, it is critical to select mAbs with low selfassociation propensity early in preclinical development to avoid later challenges in purification and formulation related to poor antibody solubility. Unfortunately, current processes to screen mAb candidates for favorable solubility behavior require purification of milligram quantities of each antibody. The © XXXX American Chemical Society
time and resources required to generate such antibody samples severely limit the number of molecules that can be analyzed. Therefore, there is a significant need for high-throughput assays that can robustly evaluate the propensity of an antibody to selfassociate. Ideally, such assays could be performed using unpurified cell culture samples that contain dilute antibodies (200 times the mAb concentration (45 μg/mL). This finding suggests that AC-SINS is weakly impacted by nonspecific proteins that do not adsorb on the capture particles. Nevertheless, we sought to further evaluate the ability of ACSINS to identify differences in mAb self-association in contaminated solutions. Thus, we asked if the presence of clarified cell culture mediawhich contains diverse contaminantswould compromise our ability to differentiate between the degree of self-association of the mAb4 mutants (Figure 8).
Figure 8. AC-SINS analysis of mAb4 variants in cell culture media. Plasmon wavelengths of mAb4 conjugates in 50% (v/v) cell culture media at pH 6. The final concentrations of gold particles and mAb variants were 8.4 × 1011 particles/mL and 45 μg/mL, respectively, and the buffer was 10 mM citrate. The capture antibody was specific for the Fc domain.
Importantly, the plasmon wavelengths of the capture particles (in the absence of mAbs) do not increase in the presence of cell culture media, revealing that the medium fails to induce nonspecific interactions. Moreover, the plasmon wavelengths of the capture particles for mAb4.3 and mAb4.4 are lower than those of the other mAb4 variants (Figure 8), as we observed for the mAbs in the absence of cell culture media (Figure 6). These findings reveal that AC-SINS is robust for assaying antibody self-interactions even in the presence of contaminants that would interfere with conventional assays of measuring antibody self-association.
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DISCUSSION The most unique aspect of AC-SINS is the use of capture antibodies to introduce specificity into self-interaction measurements that eliminates interference from non-mAb molecules ranging from proteins to low molecular weight components in cell culture media. This specificity greatly simplifies the measurements of mAb self-association in complex solutions and should enable AC-SINS analysis of unpurified mAbs as well as mAbs in serum. The stability of antibodies in serum is poorly understood yet is suspected to be an important attribute in determining the effectiveness of some therapeutic mAbs.43 Moreover, the ability of AC-SINS to assay mAb selfinteractions using dilute mAb solutions in a high-throughput format makes it well suited for use during selection of antibody candidates. Such analysis should lead to identification of G
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both the Fc and Fab regions to sample the broadest range of mAb self-interactions. The immobilization condition for the capture antibodies (pH 4.3, 2 mM acetate) was robust for all antibodies evaluated in this work and is expected to be applicable for diverse antibodies. We also find that conducting AC-SINS analysis using a range of mAb loadings (by varying the amount of immobilized capture relative to noncapture antibody) is essential for characterizing both weakly and strongly associative antibodies. Thus, we recommend that the entire range of antibody loadings (0−100% capture antibody) be evaluated for mAbs with unknown self-association propensities. In addition, we found that the mAb concentration and incubation time with the conjugates had modest but detectable effects on the AC-SINS measurements. To maximize efficiency and sensitivity, we suggest that low mAb concentrations that are sufficient to prevent nonspecific conjugate cross-linking (10−50 μg/mL absorbed on 1.68 × 1011−8.4 × 1011 gold particles/mL) and short incubation times with the target mAbs (1−2 h) be used to conduct the AC-SINS measurements. Finally, we find that the mAb purity has little impact on the AC-SINS measurements, and thus mAb solutions containing non-antibody contaminants can be used for such analysis. We expect that our AC-SINS methodology can be readily adapted to assay self-interactions for a wide range of proteins, peptides and other biomolecules via the use of alternative capture antibodies. These capture antibodies could be specific for sequence epitopes unique to target biomolecules or to common affinity tags such as biotin or polyhistidine. The extension of AC-SINS to additional well-characterized proteins will be important to further evaluate the robustness of such selfinteraction measurements and to develop a quantitative framework for interpreting the plasmon wavelength measurements. We are currently employing AC-SINS to characterize libraries of homologous antibodies to further refine AC-SINS as well as to understand how antibody sequence governs antibody self-association and solubility.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Barrett Allan for constructing the mAb4 variants. We also thank Jayapriya Jayaraman, Joseph Perchiacca, Ali Reza Ladiwala and Swarnim Ranjan for helpful comments during preparation of this manuscript. This work was supported by Eli Lilly.
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CONCLUSION AC-SINS addresses the extreme challenges of measuring mAb self-association during antibody discovery and is amenable to being conducted in parallel with antibody binding assays. We expect that the selectivity afforded by the capture antibodies will enable AC-SINS to be conducted in complex environments such as cell culture media and serum that are refractory to conventional biophysical methods. AC-SINS is also well suited for rapid characterization of purified mAbs and should be useful for formulation applications aimed at maximizing antibody solubility.
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Article
ASSOCIATED CONTENT
S Supporting Information *
Figures depicting dynamic light scattering analysis of antibody− gold conjugates, effect of human polyclonal antibody concentration on plasmon wavelength measurements, effect of mAb concentration on AC-SINS measurements, analysis of the detection sensitivity of AC-SINS, effect of incubation time on AC-SINS measurements and effect of a nonspecific protein (BSA) on AC-SINS measurements of mAb2 self-association. This material is available free of charge via the Internet at http://pubs.acs.org. H
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