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ABSTRACT: Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.
Protein Engineering Design and Selection 08/2012; 25(10):531-8. · 2.94 Impact Factor
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ABSTRACT: The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (> 100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble.
mAbs 05/2012; 4(3):319-25.
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ABSTRACT: PurposeTo develop a high-throughput cross-interaction chromatography screening method to rapidly identify antibody candidates with
poor solubility using microgram quantities of purified material.
MethodsA specific recombinant antibody or bulk polyclonal IgG purified from human serum was chemically coupled to an NHS-activated
chromatography resin. The retention times of numerous monoclonal antibodies were determined on this resin using an HPLC and
compared to the solubility of each antibody estimated by ultrafiltration.
ResultsRetention times of the antibodies tested were found to be inversely related to solubility, with antibodies prone to precipitate
at low concentrations in PBS being retained longer on the columns with broader peaks. The technique was successfully used
to screen microgram quantities of a panel of therapeutic antibodies to identify candidates with low solubility in PBS.
ConclusionsThe cross-interaction chromatography methods described can be used to screen large panels of recombinant antibodies in order
to discover those with low solubility. Addition of this tool to the array of tools available for characterization of affinity
and activity of antibody therapeutic candidates will improve selection of candidates with biophysical properties favorable
to development of high concentration antibody formulations.
Pharmaceutical Research 04/2012; 27(1):65-71. · 4.09 Impact Factor
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[show abstract]
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ABSTRACT: To develop a high-throughput cross-interaction chromatography screening method to rapidly identify antibody candidates with poor solubility using microgram quantities of purified material.
A specific recombinant antibody or bulk polyclonal IgG purified from human serum was chemically coupled to an NHS-activated chromatography resin. The retention times of numerous monoclonal antibodies were determined on this resin using an HPLC and compared to the solubility of each antibody estimated by ultrafiltration.
Retention times of the antibodies tested were found to be inversely related to solubility, with antibodies prone to precipitate at low concentrations in PBS being retained longer on the columns with broader peaks. The technique was successfully used to screen microgram quantities of a panel of therapeutic antibodies to identify candidates with low solubility in PBS.
The cross-interaction chromatography methods described can be used to screen large panels of recombinant antibodies in order to discover those with low solubility. Addition of this tool to the array of tools available for characterization of affinity and activity of antibody therapeutic candidates will improve selection of candidates with biophysical properties favorable to development of high concentration antibody formulations.
Pharmaceutical Research 11/2009; 27(1):65-71. · 4.09 Impact Factor
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Paul W Fisher,
Michael Brigham-Burke,
Sheng-Jiun Wu,
Jinquan Luo,
Jill Carton,
Kim Staquet,
Wei Gao,
Sheila Jackson, Deidra Bethea,
Cailin Chen,
Bing Hu,
Jill Giles-Komar,
Jing Yang
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ABSTRACT: Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.
Biochemical Journal 06/2005; 387(Pt 3):727-35. · 4.90 Impact Factor
