Jun Feng

The development of long-acting MC4R peptide agonists remains challenging due to rapid clearance and structural sensitivity, where conventional permanent lipidation strategies often impair receptor engagement. Here, we report the discovery and characterization of TL10, a soluble, long-acting MC4R agonist achieved via a tunable lipidation strategy that transiently masks the peptide to extend circulation while preserving activity. TL10 exhibited controlled release kinetics and a markedly prolonged plasma half-life (t1/2 ≈ 33.4 h), representing a 34-fold increase compared to setmelanotide. In diet-induced obese mice, Q3D administration of TL10 elicited sustained weight loss and appetite suppression, matching or exceeding daily setmelanotide under the tested conditions. High aqueous solubility (180 mg/mL) facilitated concentrated formulations, and a 28 day repeat-dose study indicated a favorable safety profile. Collectively, these results demonstrate that TL10 is a promising long-acting candidate with sustained pharmacological activity, providing a practical framework for the development of modification-sensitive peptides.

Acromegaly is a severe endocrine disease, and growth hormone receptor antagonist B2036 constitutes the core protein backbone of pegvisomant. Conventional B2036 production based on inclusion bodies or periplasmic expression suffers from low yield, complicated refolding, and multi-step purification. Herein, we developed a highly efficient, refolding-free bioprocess for soluble expression of B2036 in Escherichia coli using SUMO fusion technology. The entire fermentation cycle was controlled within 24 h, and fermentation optimization via response surface methodology enabled a high-yield soluble yield of 0.95 g/L. For downstream processing, an industrial-scalable, affinity-tag-free strategy using only two chromatography steps (anion-exchange and mixed-mode chromatography) was established, producing 498.47 mg/L B2036 with 97.12% purity, representing a 20-fold improvement over previous reports. The obtained B2036 showed correct sequence, precise disulfide pairing, and strong biological activity with an IC₅₀ of 0.67 nmol/L. By removing protein refolding and metal-affinity chromatography, this simplified and robust workflow remarkably improves productivity and product quality, offering a cost-effective and scalable technical platform for large-scale manufacturing of pegvisomant biosimilars. Collectively, these findings provide a valuable reference for developing efficient soluble expression and downstream purification processes of other intrachain disulfide-bonded proteins in E. coli. Graphical abstract

The biological potency of recombinant human growth hormone (rhGH) is a critical quality attribute (CQA) for ensuring clinical efficacy. However, current pharmacopoeial methods, such as the USP in vitro Nb2-11 cell proliferation assay, lack specificity as they measure lactogenic activity via prolactin receptors rather than the growth hormone receptor (GHR). In this study, we developed a highly specific and robust potency assay for rhGH by standardizing and validating a murine pro-B lymphocyte (Ba/F3) cell platform stably expressing human GHR (hGHR). While GHR-transfected cell models have been previously described for basic research, this work bridges the gap between research-grade tools and pharmaceutical quality control (QC). We established a strictly characterized monoclonal cell line to ensure the long-term genetic and functional stability essential for routine commercial testing. The engineered Ba/F3-hGHR cells exhibited a dose-dependent proliferative response exclusively triggered by hGHR signaling. Through systematic optimization of assay parameters—including cell seeding density, serum concentrations, and incubation kinetics—we established a high-performance analytical protocol. The method was rigorously validated according to ICH Q2(R2) guidelines, demonstrating superior intermediate precision (GCV 8.4–18.3%), high accuracy, and the sensitivity required to detect subtle potency shifts in degraded samples. Our findings provide a mechanism-based, validated alternative to current regulatory methods, offering a reliable approach for the accurate quantification of rhGH biological activity in biopharmaceutical manufacturing.

Background: Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, and although PD-1/PD-L1 immune checkpoint blockade has improved outcomes in some patients, therapeutic responses remain heterogeneous. Tumor-intrinsic heterogeneity within malignant epithelial populations is increasingly recognized as a critical determinant of disease progression and therapy response. Methods: Here, we constructed a comprehensive single-cell atlas of NSCLC by integrating 650,461 cells from 216 tumor and normal samples. Tumor-derived epithelial cells were reclustered to identify transcriptionally distinct subpopulations. Pseudotime analysis, functional experiments, and in vivo validation using a humanized xenograft model were performed to investigate the role of COL3A1. Results: Reclustering of tumor-derived epithelial cells revealed 25 transcriptionally distinct subpopulations. Among these, a high-risk cluster exhibited coordinated activation of epithelial–mesenchymal transition (EMT) and angiogenesis programs and was associated with poor patient survival. Within this aggressive subpopulation, Collagen type III alpha 1 (COL3A1) emerged as a tumor-intrinsic gene associated with extracellular matrix remodeling and angiogenic signaling. Pseudotime analysis indicated that COL3A1⁺ cells represent a late-stage, poorly differentiated malignant state. Functional experiments demonstrated that COL3A1 knockdown impaired NSCLC cell proliferation, migration, and invasion. Virtual knockout further suggested that COL3A1 may be associated with transcriptional programs involved in PD-L1 upstream signaling pathways, indicating a potential indirect link between tumor-intrinsic states and immune regulatory networks. Consistently, in vivo silencing of COL3A1 enhanced the antitumor efficacy of PD-L1 blockade. Conclusions: Collectively, our study identifies COL3A1 as a tumor-intrinsic gene enriched in malignant epithelial cells with mesenchymal features and a potential therapeutic target. These findings provide a rationale for exploring combinatorial strategies integrating tumor-intrinsic pathway inhibition with immune checkpoint blockade in NSCLC.

Vosoritide is a therapeutic peptide that promotes skeletal growth by targeting the NPR-B receptor and was approved in 2021 for the treatment of achondroplasia. However, its high production cost poses a considerable economic burden on patients, limiting its widespread use. This study aims to establish an efficient and cost-effective biosynthetic process for the production of Vosoritide using Escherichia coli. In this work, Vosoritide was expressed as inclusion bodies (IBs) in E. coli BL21 (DE3) via a gp55 fusion strategy. Downstream processes, including fermentation, HCl-induced acidic cleavage of IBs, pH-dependent protein precipitation, as well as the performing conditions of the ion-exchange chromatography and reverse-phase chromatography, were optimized. The analysis methods included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry. Cyclic guanosine monophosphate (cGMP) assays were conducted to assess the biological activity of the protein in NIH3T3 cells. Vosoritide was obtained at 1.3 g per liter of fermentation broth with a purity exceeding 99%. The peptide's primary structure, molecular weight, and disulfide bond integrity were also confirmed through mass spectrometry and peptide mapping. The purified Vosoritide stimulated cGMP production in NIH3T3 cells with a half-maximal effective concentration (EC₅₀) of 0.37 nmol/L. In conclusion, this study provided a scalable, high-yield production method for Vosoritide that yields Vosoritide with pharmaceutical-grade purity and activity, holding significant potential for manufacturing a cost-effective biosimilar with broader clinical accessibility. The comparison of the estimated cost of Vosoritide expression protocols between this study and previously reported studies can be found in Supporting Information ([Supplementary Table S1] [available in online version]). Received: 19 February 2025 Accepted: 02 July 2025 Article published online: 18 August 2025 © 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

Brolucizumab was the first single-chain fragment variable (scFv) antibody approved by the FDA for the treatment of age-related macular degeneration (AMD). However, the manufacturing process of brolucizumab remains rarely reported. This study aimed to explore a bioprocess for the production of brolucizumab, where it is expressed as inclusion bodies (IBs) in Escherichia coli (E. coli) BL21 (DE3) cells. In this work, IBs were initially obtained via high cell density fermentation (HCDF) at a high expression level of 30 g/L, followed by denaturation, refolding, and purification to obtain brolucizumab. The refolding parameters were systematically optimized to ensure a high yield of brolucizumab, with 413 mg of the target protein from a 1-L fermentation broth, and purity exceeding 98%. In addition, the amino acid sequence coverage and disulfide bond pairing of the protein were further verified. The results confirmed that brolucizumab has excellent structural integrity, high purity, and notable biological activity. The biosynthetic process holds significant potential for therapeutic applications of brolucizumab and provides valuable insights for the further development of additional scFv bioprocesses.