Structure-based engineering of a monoclonal antibody for improved solubility

Wu, Sheng‐Jiun; Luo, Jinquan; O’Neil, Karyn T.; Kang, James; Lacy, Eilyn R.; Canziani, Gabriela; Baker, Audrey; Huang, Maggie; Tang, Qing; Raju, T. Shantha; Jacobs, Steven; Teplyakov, A.; Gilliland, Gary L.; Feng, Yiqing

doi:10.1093/protein/gzq037

Cited by 170 publications

(195 citation statements)

References 60 publications

(74 reference statements)

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“…The involvement of CDR regions, including specific aromatic/hydrophobic residues, in RSA of antibodies has been reported in previous studies with other antibodies. 16,66,67 More specifically with mAb-C, LC 36-71 and HC 35-60, which span CDR2L and CDR2H, respectively, showed significant decreases in hydrogen exchange under RSA-promoting conditions (Fig. 7).…”

Section: Discussionmentioning

confidence: 96%

“…68 In another study, substitution of aromatic residues with non-aromatic amino acids (F99A, W100A) in the CDR3H region of the antibody caused a considerable decrease in RSA and an increase in protein solubility. 16,67 However, Fab-Fab interactions are not necessarily always responsible for such interactions between antibody molecules. For example, Nishi et al reported Fc-mediated RSA of an antibody under low ionic strength solution conditions.…”

Section: Discussionmentioning

confidence: 99%

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Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Arora

Hickey

Majumdar

et al. 2015

mAbs

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Volkin (2015) Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody, mAbs, 7:3, 525-539, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the V H domain, the constant domain (C H 2), and the hinge region (C H 1-C H 2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Arora

Hickey

Majumdar

et al. 2015

mAbs

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“…Antigen bound co-crystals are also very useful for determining mutants that are more likely to retain target binding while optimizing solubility. 7 …”

Section: Discussionmentioning

confidence: 99%

“…2–4 Successful efforts have also been made to use rational design to reduce aggregation and improve solubility by mutating hydrophobic surface regions derived from a crystal structure. 5–7 …”

Section: Introductionmentioning

confidence: 99%

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Jetha

Thorsteinson²,

Jmeian

et al. 2018

mAbs

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Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.

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“…[2][3][4] To avoid such setbacks, risk mitigation strategies increasingly center around "Quality-by-Design" (QbD) approaches, which consider developability-related issues early in the drug discovery phase and involve rationally engineering antibodies toward favorable Chemistry, Manufacturing and Control (CMC) and pharmacokinetics (PK) properties. [5][6][7] Choosing molecules that exhibit low intrinsic immunogenicity and that are robust against the formation of immunogenic degradation products can also reduce safety risks. However, the large number of different antibodies typically considered in early projects, and the small amount of each antibody that is typically available, precludes extensive experimental characterization and determination of CMC properties such as yield, chemical and physical stability.…”

Section: Introductionmentioning

confidence: 99%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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(2015) Boosting antibody developability through rational sequence optimization , mAbs, 7:3, 505-515, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and longterm stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

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Structure-based engineering of a monoclonal antibody for improved solubility

Cited by 170 publications

References 60 publications

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Boosting antibody developability through rational sequence optimization

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