Structural and In Vitro Functional Comparability Analysis of Altebrel™, a Proposed Etanercept Biosimilar: Focus on Primary Sequence and Glycosylation.

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. "Bottom-up" mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept.

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126 kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment.

Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals.

Comparability study of Rituximab originator and follow-on biopharmaceutical.

Immunglobolin G (IgG)-based biopharmaceuticals are emerging on the pharmaceuticals market due to their high target selectivity in different diseases. In parallel, a growing interest by other companies to produce similar or highly similar follow-on biologics exits, once the patent of blockbuster biotherapeutics is about to expire. In correlation to their complex structure, an analytical challenge is facing the approval of these biosimilars. Health authorities (e.g. FDA and EMA) have issued several guidelines to define critical quality attributes during manufacturing process changes. In the current study, physicochemical characterization using state-of-the-art analytics was applied to analyse intact mass, post-translational modifications (PTMs) and higher order structure of Rituximab and one of its biosimilars. Intact mass analysis, middle-up approach as well as subunit analysis revealed similar glycoforms but additional lysine variants in the biosimilar. The N-glycosylation site was confirmed for both, the originator and the biosimilar. PTMs and higher order structure were confirmed to be similar. A special focus was given to N-glycosylation due to its potential to monitor the batch-to-batch consistency and alteration during the production bioprocess. Comparison of the N-glycosylation profiles obtained from three batches of the biosimilar and the reference product showed quantitative variations, although the N-glycans were qualitatively similar. Furthermore, a head-to-head comparability of functional properties was performed to investigate the impact of glycosylation alteration and PTMs on potency within the biosimilar batches and between originator and follow-on biodrug. The data affirm that the difference is still in the acceptable range for biosimilarity.

Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase.

Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 Mut(S) strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.