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Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin

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Abstract

Purpose

To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity.

Methods

Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress. Bulk viscosity was measured by an oscillating piston viscometer.

Results

As BSA was heated at a higher concentration or for a longer time, the relative contribution, molecular weight and intrinsic viscosity of aggregate species increased. Molecular shape was evaluated from intrinsic viscosity values, and aggregates were estimated to be more asymmetric than monomer species. The presence of aggregates resulted in an increase in bulk viscosity when relative contribution of very high molecular weight species exceeded 10%.

Conclusions

For model system and conditions studied, generation of higher order aggregate species was concluded to be associated with an increase in molecular asymmetry. Elevated viscosity in the presence of aggregated species points to molecular asymmetry being a critical parameter affecting solution viscosity of BSA.

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Abbreviations

BSA:

Bovine serum albumin

DP:

Differential pressure

HMW:

High molecular weight

LALS:

Low angle light scattering

mAb:

Monoclonal antibody

mM:

Millimolar

PPI:

Protein-protein interactions

RALS:

Right angle light scattering

RI:

Refractive index

SEC:

Size exclusion chromatography

TDA:

Triple detector array

vHMW:

Very high molecular weight

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ACKNOWLEDGMENTS AND DISCLOSURES

Authors would like to thank Lauren Fontana and Rui Fang for help with some experimental aspects, and Elizabeth Zecca for critical reading of the manuscript. Generous material and financial support from Genentech, Inc., as well as Outstanding Scholar and summer fellowships from graduate school at University of Connecticut are also acknowledged.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Connecticut, U-3092, Storrs, CT, 06269, USA

    Mariya A. Pindrus, Japneet Kaur & Devendra S. Kalonia

  2. Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, CT, 06269, USA

    James L. Cole

  3. Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA

    Steven J. Shire & Sandeep Yadav

Authors
  1. Mariya A. Pindrus
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  2. James L. Cole
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  3. Japneet Kaur
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  4. Steven J. Shire
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  6. Devendra S. Kalonia
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Correspondence to Mariya A. Pindrus.

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Pindrus, M.A., Cole, J.L., Kaur, J. et al. Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin. Pharm Res 34, 2250–2259 (2017). https://doi.org/10.1007/s11095-017-2231-2

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  • DOI: https://doi.org/10.1007/s11095-017-2231-2

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