Protein Assembly in Serum and the Differences from Assembly in Buffer

Hill, John J.; Laue, Thomas M.

doi:10.1016/bs.mie.2015.06.012

Cited by 11 publications

(7 citation statements)

References 27 publications

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“…Upon addition of 1 mg/mL HSA, a peak at 8.4 S was observed (Fig. 7A), presumably resulting from direct interaction between the antibody and serum albumin, consistent with findings recently reported by Hill and Laue 53 . Increasing amounts of HSA resulted in the overall c(s 20,w ) distribution shift toward lower s-values, indicating non-ideal sedimentation behavior.…”

Section: Resultssupporting

confidence: 89%

“…The formation of complexes between fluorescently labeled protein OPG at picomolar concentrations and serum albumin as low as 1 mg/mL has been recently observed 53 . Analogous to our results, the peak corresponding to the HSA-OPG complex in HSA solutions was not detected in serum, and it was concluded that endogenous compounds in the serum compete with the OPG molecules for binding to HSA.…”

Section: Discussionsupporting

confidence: 90%

“…Similar results were obtained in the present study, where SV experiments performed with human serum alone revealed the presence of a peak with s 20,w of ∼4.0 S. For 25 nM antagonists suspended in human serum, at an optimized gain setting, approximately half of the total fluorescent signal was due to albumin. Similar contributions from albumin were reported for 10 nM anti-IgE IgG in human serum, 24 whereas in another study, the HSA signal was insignificant 53 . The intensity of the HSA-related signal can vary due to different amounts of bilirubin–HSA complex in serum from different sources or with storage times.…”

Section: Resultssupporting

confidence: 76%

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Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

Krayukhina

Noda

Ishii

et al. 2017

mAbs

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A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.

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Section: Resultssupporting

confidence: 89%

Section: Discussionsupporting

confidence: 90%

Section: Resultssupporting

confidence: 76%

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Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

Krayukhina

Noda

Ishii

et al. 2017

mAbs

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“…The applications of AUC-FDS are summarized in Table 3. In addition, AUC-FDS offers unique means for studying macromolecules in complex biological environments, such as blood serum (Demeule et al 2009;Kingsbury et al 2012;Hill and Laue 2015;Krayukhina et al 2017) or cell lysates (Kroe and Laue 2009). A number of differences in the experimental setup and technical aspects exist between AUC-FDS and conventional detection using absorbance or interference detection.…”

Section: Auc-fdsmentioning

confidence: 99%

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

2017

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Sedimentation velocity analytical ultracentrifugation (SV-AUC) coupled with direct computational fitting of the observed concentration profiles (sedimentating boundary) have been developed and widely used for the characterization of macromolecules and nanoparticles in solution. In particular, size distribution analysis by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample preparation, data acquisition, and data analysis. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.

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“…After accounting for a characteristic signal structure imposed by the optical configuration (46), it offers highly quantitative representation of the sedimentation process (46,47), and allows the detection of low picomolar concentrations of common fluorophores (32,48). This has permitted studies of labeled proteins in complex fluids such as serum (49,50) and cell lysates (29,51), and diverse studies of high-affinity interacting systems (48,(52)(53)(54)(55) with K d as low as 20 pM (48).…”

Section: Introductionmentioning

confidence: 99%

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

Chaturvedi

Zhao

Schuck

2017

Biophysical Journal

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Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems of macromolecules. It combines picomolar sensitivity with high hydrodynamic resolution, and can be carried out with photoswitchable fluorophores for multicomponent discrimination, to determine the stoichiometry, affinity, and shape of macromolecular complexes with dissociation equilibrium constants from picomolar to micromolar. A popular approach for data interpretation is the determination of the binding affinity by isotherms of weight-average sedimentation coefficients s. A prevailing dogma in sedimentation analysis is that the weight-average sedimentation coefficient from the transport method corresponds to the signal- and population-weighted average of all species. We show that this does not always hold true for systems that exhibit significant signal changes with complex formation-properties that may be readily encountered in practice, e.g., from a change in fluorescence quantum yield. Coupled transport in the reaction boundary of rapidly reversible systems can make significant contributions to the observed migration in a way that cannot be accounted for in the standard population-based average. Effective particle theory provides a simple physical picture for the reaction-coupled migration process. On this basis, we develop a more general binding model that converges to the well-known form of s with constant signals, but can account simultaneously for hydrodynamic cotransport in the presence of changes in fluorescence quantum yield. We believe this will be useful when studying interacting systems exhibiting fluorescence quenching, enhancement, or Förster resonance energy transfer with transport methods.

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Protein Assembly in Serum and the Differences from Assembly in Buffer

Cited by 11 publications

References 27 publications

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield

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