Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio.

Generation and utilization of endostatin-sensitive cell lines for assessing the biological activity of endostatin.

Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult.

Integrating ultra-high-performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in-depth characterization of complex fusion proteins.

Rationale: Fc-fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods.

Methods: Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra-high performance liquid chromatography (UHPLC-MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in-depth fusion protein characterization.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b.

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b.

Epigallocatechin gallate attenuates tumor necrosis factor (TNF)-α-induced inhibition of osteoblastic differentiation by up-regulating lncRNA TUG1 in osteoporosis.

Promoting osteoblast proliferation and differentiation contributes to the prevention and clinical treatment of osteoporosis. This study was to investigate the effect and mechanism of epigallocatechin gallate (EGCG) on tumor necrosis factor (TNF)-α-caused inhibition of osteoblastic differentiation. First, we cultured mouse embryo osteoblast precursor cells (MC3T3-E1) and induced by TNF-α (0, 2.

Development of an Adeno-Associated Virus-Vectored SARS-CoV-2 Vaccine and Its Immunogenicity in Mice.

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus.

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF.

Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality.

Development and Validation of a Reporter-Cell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc.

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc).

Anti-Tumor Activity and Pharmacokinetics of AP25-Fc Fusion Protein.

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time is only about 50 minutes, which limits its clinical application.

[Characterization and comparison of interferon reference standards using UPLC-MS].

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample.

Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo.

Objective: A recombinant antitumor/antiviral protein (Novaferon, Nova) is a new type of interferon, which is produced by artificial design technology combining DNA-shuffling and High Throughput Screening (HTS).

Methods: The in vitro biological activities, such as anti-tumor activity and antiviral activity of Nova and recombinant human interferon alpha-2b (rhIFN-α2b) was performed; in vivo anti-tumor activity in nude mice was also tested. Flow cytometry, histo-pathological analysis including HE staining and immunohistochemistry, and surface plasmon resonance assay were performed to investigate the underlying mechanisms analysis.

[Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS].

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3.