Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation

Wright, Robert T.; Hayes, David; Stafford, Walter F.; Sherwood, Peter J.; Correia, John J.

doi:10.1016/j.ab.2018.04.002

Cited by 31 publications

(36 citation statements)

References 54 publications

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“…Human adult serum contains high concentrations of serum albumin (∼30–40 mg/mL) in addition to IgGs (∼10–15 mg/mL) that might impact these results. However, work by Wright et al, using different mAb tracers in mixtures that include albumin show weak IgG:IgG interactions that are consistent with these findings …”

Section: Discussionsupporting

confidence: 59%

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Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

et al. 2018

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Weak protein-protein interactions may be important to binding cooperativity. A panel of seven fluorescently labeled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing concentrations of unlabeled IgGs of identical, similar, and different backgrounds. Weak IgG::IgG attractive interactions were detected and characterized by global analysis of the hydrodynamic nonideality coefficient, k . The effects of salt concentration and temperature on k suggest the interactions are predominantly enthalpic in origin. The interactions were found to be variable in strength, affected by both the variable and constant regions, but indiscriminate with respect to IgG subclass. Furthermore, weak attractive interactions were observed for all the mAbs with freshly purified human poly-IgG. The universality of the weak interactions suggest that they may contribute to effector function cooperativity in the normal immune response, and we postulate that the generality of the interactions allows for a broader range of epitope spacing for complement activation. These studies demonstrate the utility of analytical ultracentrifuge fluorescence detection in measuring weak protein-protein interactions. It also shows the strength of global analysis of sedimentation velocity data by SEDANAL to extract hydrodynamic nonideality k to characterize weak macromolecular interactions.

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Section: Discussionsupporting

confidence: 59%

“…However, work by Wright et al, using different mAb tracers in mixtures that include albumin show weak IgG:IgG interactions that are consistent with these findings. 43…”

Section: Discussionmentioning

confidence: 99%

Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

et al. 2018

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“…The slope of the line reflects the thermodynamic nonideality k s . With increasing temperature, the slope of the line decreases corresponding to smaller apparent k s values caused by the presence of oligomers (28). By 30 C, the slopes are negative corresponding to negative k s values and consistent with the presence of stronger concentration-dependent oligomer formation.…”

Section: R H Of Elp and Dox-elp Increases As A Function Of Concentratmentioning

confidence: 64%

“…To see this figure in color, go online. , the sedimentation coefficient at zero concentration, and the slopes correspond to ks, the apparent hydrodynamic nonideality (26,28). To see this figure in color, go online.…”

Section: Discussionmentioning

confidence: 99%

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Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides

Zai-Rose

West

Krämer

et al. 2018

Biophysical Journal

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The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent b-turn structures. Labeling with Dox further enhances b-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Doxlabeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a À11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets.

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Analysis of Macromolecular Size Distributions in Concentrated Solutions

Chaturvedi,

Schuck

2024

Chemistry Methods

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The solution state of macromolecules in concentrated solutions impacts fields ranging from cell biology, to colloid chemistry and engineering of protein pharmaceuticals. Dependent on the interplay between repulsive and weakly attractive forces, proteins may exhibit oligomerization, aggregation, crystallization, liquid‐liquid phase separation, or the formation of multiprotein complexes. The particle size‐distribution is a key characteristic, but difficult to determine when interparticle distances are on the order of their size and macromolecular motion is coupled through hydrodynamic interactions. Here we extend sedimentation velocity analytical ultracentrifugation to measure macromolecular size distributions under these conditions: We apply results from statistical fluid mechanics for the concentration‐dependence of hindered settling and diffusion, embedded in a mean‐field approximation that can resolve coupled sedimentation and diffusion processes of different sized species given experimental sedimentation data. This is combined with a description of transient optical aberrations from lensing in the refractive index gradients associated with sedimentation boundaries (Wiener skewing). We demonstrate this approach in the application to protein solutions with macromolecular volume fractions up to ≈10 %, for example, resolving monomers and dimers of albumin at 140 mg/ml. This enables size‐distribution analysis of proteins at concentrations of therapeutic antibody formations and close to physiological concentration in serum and cells.

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Characterization of therapeutic antibodies in the presence of human serum proteins by AU-FDS analytical ultracentrifugation

Cited by 31 publications

References 54 publications

Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

Weak IgG self‐ and hetero‐association characterized by fluorescence analytical ultracentrifugation

Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides

Analysis of Macromolecular Size Distributions in Concentrated Solutions

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