Various preparations of follicle-stimulating hormone (FSH) are commercially available; however, they differ in glycoforms composition and purity owing to their respective sources. Additional ...chemical/physical changes can also be introduced during manufacturing and can impact their biological activity (biopotency), which is routinely assessed using an in vivo bioassay (Steelman-Pohley). This study aimed to determine whether an in vitro bioassay could assess biopotency by distinguishing between r-hFSH chemical/physical variants with similar ability to the in vivo bioassay. The specific activity (units of biological activity per mg of product) of variants of r-hFSH generated through enrichment (acidic/basic), stress (oxidative/acidic pH) and enzymatic treatment (desialylation and desialylation/degalactosylation) was compared using the in vivo and in vitro bioassays. The in vitro bioassay reliably detected potential chemical/physical modifications in r-hFSH variants that may impact biopotency. Overall, the methods demonstrated a comparable ability to detect changes in specific activities due to chemical/physical differences in r-hFSH variants. These data indicate that the in vitro bioassay is suitable to replace the in vivo bioassay.
Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and ...manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.
Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs ...have emerged. Many research groups have focused their attention towards o-nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems.
Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile ...dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses
. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test;
< 0.05,
= 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10
-1 × 10
ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells
. Intracellular cAMP production, Ca
increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test;
< 0.001;
= 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC
range = 12 ± 0.9-24 ± 1.7 ng/ml; β-arrestin 2 EC
range = 140 ± 14.1-313 ± 18.7 ng/ml; Kruskal-Wallis test;
≥ 0.05;
= 4). Kinetics analysis revealed that intracellular Ca
increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test;
< 0.05;
= 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC
s were demonstrated (Kruskal-Wallis test;
> 0.05;
= 5). Apart from preparation-specific intracellular Ca
increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.
Rationale
We show evidence of cysteinylation on Cys252 of recombinant human p40 subunit of interleukin 12 (IL‐12). This was reported in 1996. However, no paper detailing this concept has been ...published yet. Our paper reports the quantification of Cys252 cysteinylation as well as the full disulfide bridges assignment by nonreducing peptide mapping using mass spectrometry (MS) detection.
Methods
Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra‐high‐performance liquid chromatography–MS analysis was performed on a Waters quadrupole time‐of‐flight Xevo G2‐XS mass spectrometer acquiring data in MSE mode. MS data were processed using Expressionist MS Refiner 13.5 (Genedata).
Results
Scouting experiments were performed using two batches of drug substance. An in‐depth study of the LC tandem mass spectrometry profiles revealed the presence of additional species related to “free” Cys252; this cysteine residue was also detected in its S‐cysteinylated and S‐homocysteinylated forms. This result is consistent with that reported in literature so far. The relative abundance of overall “cysteinylated” species resulted in the range between 46% and 36%, which has also been confirmed using orthogonal techniques such as Ellman’s assay.
Conclusions
Our data clearly demonstrate that the free cysteine (Cys252) on the p40 subunit of recombinant IL‐12 is also present in its cysteinylated and homocysteinylated forms at a considerable rate. Our observations, although based on results obtained on an IL‐12‐derived fusion protein, are consistent with the current literature.
There is an increasing interest in the generation of Fc‐fusion molecules to exploit the effector functions of Fc and the fusion partner, towards improving the therapeutic potential. The Fc‐fusion ...molecules have unique structural and functional attributes that impart various advantages. However, the manufacturing of Fc‐fusion molecules possesses certain challenges in the biopharmaceutical development. The fusion of unnaturally occurring two or more domains in a construct can pose problems for proper folding and are prone to aggregation and degradation. Reshuffling of disulfide bridges represents a posttranslational event that affects folding. This can play a critical role in the correct structure of a molecule and leads to structural heterogeneity in biotherapeutics; it may also impact the in vivo biological activities, safety, and efficacy of the biopharmaceutical. Our work presents an investigation case of a doublet band, as observed only in nonreducing sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis (SDS‐PAGE) for a bi‐specific, N‐ and C‐terminal Fc‐fusion molecule. Other characterization and orthogonal methods from the analytical panel did not indicate the presence of two distinct species, including the orthogonal CE‐SDS (Caliper Lab Chip GXII). Therefore, it was necessary to determine if the phenomenon was an analytical artifact or a real variant of our Fc‐fusion molecule. With the comprehensive mass spectrometry‐based characterization, we were able to determine that the doublet band was related to the reshuffling of one disulfide bridge in one of the fused domains. Our work illustrates the application of nonreducing peptide mapping by mass spectrometry to characterize and identify disulfide variants in a complex N‐ and C‐terminal Fc‐fusion molecule, and further adoption to monitor the disulfide structural variants in the intermediate process samples to drive the manufacturing of a consistent product with the desired quality attributes.
A key aspect that must be supervised during the development of a biotherapeutic is the presence of elemental impurities in the final drug product: they must be quantified as to ensure that their ...concentrations does not affect patients’ safety. Regulatory guidelines such as ICH Q3D provides Permitted Daily Exposure (PDE) limits for those impurities considered having a higher potential safety risk. However, one of the limits of such PDE values is that they account for the safety risk, while alterations of certain Quality Attributes (QA) of a biologic may also take place. In order to understand how certain impurities could affect not only the safety of patients, but also the physicochemical properties of biotherapeutics, here we present a study in which we examined how four commonly observed elemental impurities could impact the QAs of a Fc-fusion protein, under normal storage conditions and after six weeks of incubation at +25 °C and +40 °C. The molecule was indeed treated with increasing concentrations of Ni2+, Cu2+, Zn2+ and Fe3+ and the potential changes in conformation, oxidation, aggregation, and fragmentation were monitored. Our data suggest that keeping the levels of these impurities under the safety threshold limits does not guarantee the product quality. While nickel and zinc slightly altered the physicochemical properties of our Fc-fusion protein, iron and copper appeared to be more harmful for the QAs stability. Indeed, these latter elements might cause significant alterations of the product quality such as to potentially alter its efficacy.
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Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase ...both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.
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