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Research ArticleTechnology/Application

Characterizing the Overall Derivatization of Conjugated Oligomeric Proteins

Michaela Wendeler, Wei Zhao, Christopher Thompson, Manuel Baca, Dhanesh Gadre, Xiangyang Wang and Harry Yang
PDA Journal of Pharmaceutical Science and Technology March 2017, 71 (2) 115-126; DOI: https://doi.org/10.5731/pdajpst.2016.006510
Michaela Wendeler
2Department of Purification Process Sciences, and
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Wei Zhao
1Department of Nonclinical Biostatistics,
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Christopher Thompson
2Department of Purification Process Sciences, and
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Manuel Baca
3Department of Antibody Discovery and Protein Engineering, MedImmune, LLC, Gaithersburg, MD, USA
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Dhanesh Gadre
2Department of Purification Process Sciences, and
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Xiangyang Wang
2Department of Purification Process Sciences, and
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Harry Yang
1Department of Nonclinical Biostatistics,
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  • For correspondence: YangH@MedImmune.com
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Abstract

The analysis and accurate quantitation of bioconjugations proves challenging in the case of oligomeric proteins, especially when the size of the molecule or the nature of the conjugate do not allow the analysis of the intact protein under native conditions. In this case, analytical methods are frequently applied that result in a dissociation of non-covalently linked subunits. This limits the analysis to a description of individual subunits, thereby obscuring the accurate characterization of the overall functionalization. This situation is frequently encountered in the biopharmaceutically important case of protein PEGylation, as the biophysical properties of the PEG polymer generally make analysis and accurate quantitation for a protein with multiple conjugation sites challenging under native conditions. In this work we present a statistical measure for deriving the overall functionalization of an oligomeric protein from the data obtained from readily accessible assays that cause non-covalently associated subunits to dissociate. This approach is broadly applicable for the characterization and optimization of bioconjugation reactions for multimeric biomolecules. It should also be highly valuable for the accurate description of composition and manufacturing consistency of conjugated biotherapeutics in regulatory filings.

LAY ABSTRACT: Conjugated proteins are an important class of biopharmaceuticals. For these molecules, successful drug development requires accurate methods for the quantitative characterization of protein conjugation. This task is particularly challenging in the case of proteins consisting of several, non-covalently linked subunits, especially when the size of the protein or nature of the conjugate do not allow for analysis of the intact oligomeric molecule. Many of the analytical methods used to characterize these conjugates, such as reverse phase high-performance liquid chromatography, cause the individual subunits to dissociate, making it difficult to fully understand quality attributes at the native oligomeric level. We present a method to accurately quantify the overall conjugation of an oligomeric protein in these cases when readily available assays describe only individual subunits. This should be highly valuable for process optimization and to correctly characterize the conjugated biopharmaceutical in interactions with regulatory agencies.

  • Bioconjugation
  • Oligomeric protein
  • PEGylation
  • Multinomial distribution
  • © PDA, Inc. 2017
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PDA Journal of Pharmaceutical Science and Technology: 71 (2)
PDA Journal of Pharmaceutical Science and Technology
Vol. 71, Issue 2
March/April 2017
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Characterizing the Overall Derivatization of Conjugated Oligomeric Proteins
Michaela Wendeler, Wei Zhao, Christopher Thompson, Manuel Baca, Dhanesh Gadre, Xiangyang Wang, Harry Yang
PDA Journal of Pharmaceutical Science and Technology Mar 2017, 71 (2) 115-126; DOI: 10.5731/pdajpst.2016.006510

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Characterizing the Overall Derivatization of Conjugated Oligomeric Proteins
Michaela Wendeler, Wei Zhao, Christopher Thompson, Manuel Baca, Dhanesh Gadre, Xiangyang Wang, Harry Yang
PDA Journal of Pharmaceutical Science and Technology Mar 2017, 71 (2) 115-126; DOI: 10.5731/pdajpst.2016.006510
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Keywords

  • Bioconjugation
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  • PEGylation
  • Multinomial distribution

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