Purpose
Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future
in vivo
studies.
Methods
Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum.
Results
Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum.
Conclusions
The developed
in vitro
IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future
in vivo
studies with ICs.
The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.
The biological activity of the glycoprotein hormone erythropoietin (EPO) is dependent mainly on the structure of its N‐linked glycans. We aimed to readily attach defined N‐glycans to EPO through copper‐catalyzed azide alkyne cycloaddition. EPO variants with an alkyne‐bearing non‐natural amino acid (Plk) at the N‐glycosylation sites 24, 38, and 83 were obtained by amber suppression followed by protein purification and refolding. Click conjugation of the alkynyl EPOs with biantennary N‐glycan azides provided biologically active site‐specifically modified EPO glycoconjugates.
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