As patents for biopharmaceuticals that have been in use for decades have expired, many pharmaceutical companies are developing biosimilars. Biosimilars should be comprehensively evaluated by applying various analytical techniques for the similarities and differences with the reference product. In particular, since therapeutic glycoproteins have very complicated structure, dozens of analytical methods should be developed and applied to develop a single biosimilar product. The therapeutic glycoprotein characterization consists of physicochemical characterization to analyze primary structure, glycosylation, physicochemical properties (electrophoresis, chromatographic, spectroscopic characteristics) and immunochemical properties, and biological characterization. Individual analytical methods should be developed as sensitive and reproducible methods based on sound scientific principles.The results of characterization study using these analytical methods can be complementary and consistent with each other in order to comprehensively show the characteristics of therapeutic glycoprotein. In this study, N-glycan analytical method to evaluate the most important quality attribute of therapeutic glycoprotein was developed and a comprehensive characterization study to compare the proposed biosimilar, LBDE with reference product, $NESP^®$ was performed.
N-glycans of therapeutic glycoproteins are critical quality attributes that should be monitored throughout all stages of biopharmaceutical development and manufacturing life cycle in industry. To reduce the time for sample preparation and variations in analytical results, we developed the N-glycan analysis method including improved 2-aminobenzoic acid (2-AA) labeling. Using this analytical method, 15 major 2-AA labeled N-glycans of $Enbrel^®$ were separated into single peaks in hydrophilic interaction chromatography mode and therefore could be quantitated. 2-AA labeled N-glycans were also highly compatible with in-line quadrupole time-of-flight mass spectrometry (Q-TOF MS) for the structural identification. The structures of 15 major and 18 minor N-glycans were identified from their mass values determined by Q-TOF MS. Furthermore, the structures of 14 major N-glycans were confirmed by interpreting the MS/MS data of each N-glycan. This analytical method was also successfully applied to neutral N-glycans of $Humira^®$ and highly sialylated N-glycans of $NESP^®$. Furthermore, the analysis data of $Enbrel^®$ that were accumulated for 2.5 years demonstrated high-level consistency of this analytical method. Taken together, a wide repertoire of N-glycans of therapeutic glycoproteins can be analyzed with high efficiency and consistency using improved 2-AA labeling based N-glycan analysis method.
For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization.To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and $NESP^®$, its originator, we performed a comprehensive physicochemical and biological characterization study.Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography-mass spectrometry (LC-MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) was evaluated using rats through two injection routes.Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and $NESP^®$ have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with $NESP^®$. For both LBDE and $NESP^®$, a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with $NESP^®$ in the binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. PK parameters of LBDE were in good agreement with those of $NESP^®$.LBDE shows high similarity to $NESP^®$with regard to structure and function.
In conclusion, N-glycan analysis method based on improved 2-aminobenzoic acid labeling was successfully developed and optimized for three therapeutic glycoproteins ($Enbrel^®$, $Humira^®$ and $NESP^®$). 20 physicochemical characterization and 4 biological characterization methods were developed and applied to fully understand and compare the quality attributes of proposed biosimilar, LBDE and reference product, $NESP^®$. And LBDE showed high similarity with $NESP^®$.