title: Characterizing the Overall Derivatization of Conjugated Oligomeric Proteins.
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.
authors.affiliation: Department of Purification Process Sciences, and.
authors.forename: Michaela
authors.initials: M
authors.lastname: Wendeler
authors.affiliation: Department of Nonclinical Biostatistics.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Department of Purification Process Sciences, and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Thompson
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune, LLC, Gaithersburg, MD, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Baca
authors.affiliation: Department of Purification Process Sciences, and.
authors.forename: Dhanesh
authors.initials: D
authors.lastname: Gadre
authors.affiliation: Department of Purification Process Sciences, and.
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Nonclinical Biostatistics, YangH@MedImmune.com.
authors.forename: Harry
authors.initials: H
authors.lastname: Yang
mesh_terms: D001665:Binding Sites; D002626:Chemistry, Pharmaceutical / Q000379:methods* / Q000706:statistics & numerical data; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D008301:Maleimides / Q000737:chemistry; D015233:Models, Statistical; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D008301:Maleimides; D011994:Recombinant Proteins; C043592:maleimide; D011092:Polyethylene Glycols
keywords: Bioconjugation; Multinomial distribution; Oligomeric protein; PEGylation