O-glycosylation is a common post-translational modification on extracellular and secreted proteins driving biochemical and biophysical interactions at the cell surface. Glycosylation affects drug immunogenicity, efficacy, and clearance, making it a critical attribute of biotherapeutics. Unlike N-linked glycans, O-linked glycans are difficult to characterize because there is no consensus sequence for glycosylation sites on the polypeptide and a universal enzyme to release O-glycans from proteins. To overcome these hurdles, O-glycan analysis and localization require an appropriate and well-validated approach, particularly for recombinant human follicle stimulating hormone-C-terminal peptide (rhFSH-CTP). FSH-CTP consists of a native FSH α/β subunit fused with the C-terminal fragment of a human chorionic gonadotropin (hCG) β subunit, which is heavily O-glycosylated. However, few FSH-CTP O-glycosylation identification methods exist. Thus, we developed a characterization method for the O-linked glycan profile and glycosylation sites of rhFSH-CTP. By means of O-glycan profiling, we identified predominantly core 1-based structures with good reproducibility. For site-specific localization, the O-glycopeptidase OpeRATOR, used with sialidase, helped identify O-glycosylated peptides. Electron transfer/higher-energy collision dissociation (EThcD), combined with OpeRATOR, identified all six glycosylation sites. This approach improves quality control for rhFSH-CTP biosimilars and other CTP-fusion proteins, contributing to the development of standardized O-glycan identification methods.

PEGylated recombinant human growth hormone (PEG-rhGH) has garnered significant interest in growth hormone research due to its prolonged half-life and improved patient compliance. An accurate evaluation of its biological activity is critical for ensuring the quality of PEG-rhGH-based therapeutics. In this study, we established an in vitro bioactivity assay using a reporter gene method based on the HepG2/IGF-1 cell line. Key assay parameters, including the initial concentration of PEG-rhGH, serial dilution ratios, cell density, and incubation time, were systematically optimized to generate robust dose–response curves. The assay demonstrated high sensitivity, precision, and reproducibility across multiple batches of PEG-rhGH. The validation results showed an excellent correlation with traditional in vivo animal studies and the Nb2-11 cell proliferation assay, highlighting its suitability for quality control. Furthermore, we developed an ion exchange chromatography (IEC) method to separate five positional isomers of PEG-rhGH, revealing significant differences in bioactivity depending on the PEG modification site. This study demonstrates that the optimized reporter gene assay is not only effective for quality control of PEG-rhGH but also serves as a valuable tool for evaluating and optimizing PEGylated long-acting growth hormone therapeutics.

Glucagon-like peptide-2 (GLP-2) is a gut hormone that plays a pivotal role in regulating intestinal epithelial cell growth and function, making it a promising therapeutic agent for intestinal damage and bone-related diseases. Nonetheless, the therapeutic potential of GLP-2 is substantially diminished due to its inactivation by dipeptidyl peptidase 4 (DPP-4). In recent years, advancements have been made in developing dipeptidyl peptidase 4 (DPP-4) resistant GLP-2 analogues with an extended half-life. The murine model with extensive experimental bowel resection maintained on parenteral nutrition has been used for assessing the physiology and pharmacology of GLP-2, and for the preclinical validation of GLP-2 analogues. However, it possesses certain limitations, such as complex procedure, considerable variability, and time-consuming nature. Consequently, there is a pressing need for the development of a cell-based bioassay to assess GLP-2 analogues. Here, we successfully developed a mechanism-of-action (MOA)-reflective cell-based reporter gene assay (RGA), utilizing a stable HEK293 cell line expressing the GLP-2 receptor and a luciferase reporter gene. This innovative approach allows for precise quantification of the potency of GLP-2 analogues. The RGA demonstrated good accuracy, linearity, precision, and specificity, with potential applications in stability testing, drug screening, and therapeutic monitoring of GLP-2 analogues. Moreover, RNA sequencing reveals the multi-target regulatory effect of GLP-2 analogues. The establishment of this RGA provides a valuable tool for evaluating the potency of GLP-2 analogues and the screening of potential therapeutic drugs targeting to GLP-2 receptor.

Inconsistent conclusions on the cellular uptake of recombinant human β-glucocerebrosidase (rhGCase) for Gaucher disease stem from a fundamental limitation of existing methods: their inability to generate complete and reliable dose–response curves. This critical flaw, stemming from susceptibility to various experimental variables, prevents accurate potency comparison across different rhGCase products. To address this, we developed a robust bioassay using CHO-K1 cells stably expressing the human macrophage mannose receptor (hMMR). Our method quantifies uptake by measuring the enzymatic activity of internalized rhGCase and consistently produces a classic sigmoidal dose–response curve. Comprehensive validation and mechanistic studies, including inhibition experiments with mannose, fucose, and mannose-6-phosphate, confirmed that uptake is specifically mediated by hMMR, with successful enzyme transport to endosomes/lysosomes. Applying this assay to three commercial products yielded results contrary to prior literature: imiglucerase demonstrated superior uptake activity to velaglucerase alfa. The proposed method represents a significant improvement over existing assays, providing a more accurate and reproducible means to evaluate cellular uptake bioactivity, which is crucial for the quality control of rhGCase therapeutics.

To develop a cell-based in vitro thyroid-stimulating hormone (TSH) biological activity assay that can simulate in vivo pharmacodynamic mechanisms, we constructed two HEK293-TSHR cell lines based on two main cell signaling pathways (Gαs-cAMP-PKA and Gαq/11-PLC-Ca²⁺) that TSH depends on for its in vivo physiological function. These cell lines stably expressed the luciferase reporter driven by the cAMP response element (CRE) and nuclear factor of activated T cells (NFAT) response element, and two reporter-gene assays (RGAs) were correspondingly established and validated. The two transgenic genes could measure signals produced from the simulation of the in vivo effects of TSH from the Gαs-cAMP and Gαq/11-PLC pathways after TSH activation. TSH showed a good dose–response relationship in these two cell lines and conformed to the four-parameter model. We optimized the critical experimental parameters of these two methods and performed comprehensive methodological validation according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, the Chinese Pharmacopoeia, and the United States Pharmacopoeia. The two methods showed good specificity, accuracy, precision, and linearity and can be used to aid in assessments of the biological activity of TSH drugs, product characterization, final product release, stability studies, and comparability studies for biosimilar applications.