host-cell-protein-analysis-using-enrichment-beads-coupled-with

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

knockdown-fam83a-to-verify-its-role-cervical-cancer-cell-growth

Knockdown of <em>FAM83A</em> to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps ...

Regeneron Pharmaceuticals Inc.

Yangtze University

Polysorbates (PSs), including PS20 and PS80, are non-ionic surfactants widely used in the pharmaceutical industry to enhance drug solubility and stability. Despite their wide application, PSs are prone to degradation by either hydrolysis or oxidation in drug formulations during storage; therefore, a PS characterization method assessing protein products is needed for stability testing and for understanding the degradation pathway. In this article, we detail our protocol for sample preparation for forced degradation study and our instrumentation setup for PS profiling and quantitation in protein samples. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for forced degradation of polysorbate in protein samples Basic Protocol 2: Two-dimensional liquid chromatography coupled with charged aerosol detector or mass spectrometry to analyze polysorbate degradation.

The Characterization of Polysorbate.

Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination.

Analysis of Host Cell Proteins in Monoclonal Antibody Therapeutics Through Size Exclusion Chromatography.

Host cell proteins (HCPs) are process-related impurities that remain in therapeutic protein -at trace levels. HCPs must be closely monitored because they may be detrimental to drug product quality. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for detecting individual HCPs; however, HCP-derived peptides can be four to ten orders of magnitude less abundant than therapeutic protein-derived peptides in drug products, thus posing a major challenge in LC-MS detection. We previously demonstrated that low abundant HCPs can be enriched several hundreds fold through ProteoMiner. This study further improved the degree of enrichment by coupling limited digestion to ProteoMiner technology (PMLD). HCPs with low abundance were enriched 7694-fold, thus enabling detection of HCPs at concentrations as low as 0.002 ppm. A total of 850 HCPs were detected with high confidence from a NIST monoclonal antibody preparation, a number 40% higher than previously reported.

Ultrasensitive method for profiling host cell proteins by coupling limited digestion to ProteoMiner technology.

Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.

Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics.

Cervical cancer (CC) is the most common malignant tumor in females. Although persistent high-risk human papillomavirus (HPV) infection is a leading factor that causes CC, few women with HPV infection develop CC. Therefore, many mechanisms remain to be explored, such as aberrant expression of oncogenes and tumor suppressor genes. To identify promising prognostic factors and interpret the relevant mechanisms of CC, the RNA sequencing profile of CC was downloaded from the Cancer Genome Atlas and the Gene Expression Omnibus databases. The GSE63514 dataset was analyzed, and differentially expressed genes (DEGs) were obtained by weighted coexpression network analysis and the edgeR package in R. Fifty-three shared genes were mainly enriched in nuclear chromosome segregation and DNA replication signaling pathways. Through a protein-protein interaction network and prognosis analysis, the kinesin family member 14 (KIF14) hub gene was extracted from the set of 53 shared genes, which was overexpressed and associated with poor overall survival (OS) and disease-free survival (DFS) of CC patients. Mechanistically, gene set enrichment analysis showed that KIF14 was mainly enriched in the glycolysis/gluconeogenesis signaling pathway and DNA replication signaling pathway, especially in the cell cycle signaling pathway. RT-PCR and the Human Protein Atlas database confirmed that these genes were significantly increased in CC samples. Therefore, our findings indicated the biological function of KIF14 in cervical cancer and provided new ideas for CC diagnosis and therapies.

Dysregulation of KIF14 regulates the cell cycle and predicts poor prognosis in cervical cancer: a study based on integrated approaches.

Cervical cancer (CC) is one of the most common gynecological tumors worldwide. Several studies have reported that circular RNAs (circRNAs) play important roles in various types of diseases, including cancer. Thus, the present study aimed to investigate the role of circRNA_0000285 in CC development. Dual-luciferase reporter and RNA pull-down assays were performed to verify the binding region between circRNA_0000285 and miR-654-3p. The expression levels of circRNA_0000285 and miR-654-3p were analyzed in CC and the corresponding normal tissues, as well as in SiHa, HeLa, and NC104 cells using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, the effect of circRNA_0000285 inhibition on cell viability, apoptosis, and the expression of apoptosis-related markers was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), flow cytometry, and Western blotting assays, respectively. The results verified that miR-654-3p directly targeted circRNA_0000285 expression. circRNA_0000285 was overexpressed and miR-654-3p expression was downregulated in CC tissues and cells compared to that in control. Moreover, circRNA_0000285 knockdown suppressed the viability and promoted the apoptosis of CC cells, which was accompanied by the downregulated and upregulated expressions B-cell lymphoma-2 () and Bcl-2 associated X (), respectively. The ratio of Bax/Bcl-2 levels also increased following circRNA_0000285 knockdown. However, these findings were abrogated after miR-654-3p inhibitor treatment. Hence, circRNA_0000285 knockdown suppressed cell viability and promoted apoptosis by targeting miR-654-3p in CC.

Sisi Zhang

Analytical Chemistry

Regeneron Pharmaceuticals Inc.

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity