Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Biosimilar | Biosimilarity Assessment
Authors: Jukyung Kang, University of Michigan (Main Author, Presenting Author); Yuwei Tian, University of Michigan; Vivekanandan Subramanian, University of Michigan; Ilker Sen, Protein Metrics; Rose Ackermann, University of Michigan; Brandon Ruotolo, University of Michigan; Anna Schwendeman, University of Michigan
Presenting Author: Jukyung Kang
Purpose: The purpose of this study is to demonstrate physico-chemical biosimilarity between a potential biosimilar filgrastim and its reference, Neupogen®, using a variety of analytical methods and techniques. A biosimilar is a biological product that has no clinically meaningful difference to its reference in terms of safety, purity and potency. Due to patent expirations and approval of the first biosimilar in US, the opportunity for biosimilar approval has increased. Filgrastim is a granulocyte-colony stimulating factor (G-CSF), 175 amino acid protein manufactured by recombinant process. Here we examine the analytical comparability between innovator Neupogen and a filgrastim biosimilar, testing inherent variability due to the differences in manufacturing process and scaling between the innovator and the biosimilar.
Methods: Six lots of Neupogen® and biosimilar were used for physico-chemical analysis. Samples were analyzed for aggregation and post-translational modification (PTM) by size exclusive chromatography (SEC) and LC-MS/MS peptide analysis following Glu-c digestion. Data was processed using Protein Metrics Byologic® software. 2D 1H-15N HSQC NMR data, using concentrated samples, were collected for 84 h at 15&[deg]C, and Ion mobility mass spectrometry (IM-MS) spectra, using two step buffer exchanged samples to 100 mM ammonium acetate, were obtained to assess higher-order structure. Reverse phase chromatography was performed to analyze sample purity, detecting oxidized and deamidated variants.
Results: The primary sequence of Neupogen® and its biosimilar are identical. Processing software shows 100% coverage. In terms of PTM, both products are analytically similar with the exception of deamination of glutamine 12. Further research is needed to show the effect of this change. 1D NMR spectra reveals high structural similarity. Notably, the amide back bone region does not show any differences (between 6-10 ppm). 2D NMR spectra shows that amide peaks in the spectra were well dispersed from 6.3 to 9.75 ppm, indicating the protein is well-folded. Statistical comparison of both 2D NMR data proves that there is no detectable difference between Neupogen® and its biosimilar. IM-MS data show Neupogen® and its biosimilar to have highly similar fragmentation and aggregation populations, also showing comparable unfolding patterns.
Conclusion: Based on comprehensive analysis with the state-of-the-art techniques, Neupogen® and its biosimilar were highly similar in regards to physico-chemical properties.
See attached abstract pdf for images.