Host Cell Proteins Research Articles (Page 1)

Host cell protein detection via surfactant-assisted acid precipitation.

Host cell protein quantitation by LC-MS. Experimental demonstration, qualification, and comparison of methods in USP 1132.1.

Major vault protein inhibits CSFV replication by promoting cell apoptosis.

Improving recombinant antibody production using FcBAR: An in situ approach to detect and amplify protein-protein interactions.

The facultative intracellular pathogen Legionella pneumophila, which causes Legionnaires' disease, translocates over 300 effector proteins into the host cell. By hijacking numerous host cellular signaling pathways, these effectors promote bacterial survival and growth. One of the effector proteins, LegU1, is a F-box containing protein that binds to the host cell protein Skp1 to form a Skp1-Cullin-F-box protein (SCF) complex conferred E3 ubiquitin ligase activity. However, the role of LegU1 during L. pneumophila infection is incompletely known. Here, we demonstrate that LegU1 participates in modulation of the host BiP, an important endoplasmic reticulum chaperone that functions as both a master regulator and target protein of the unfolded protein response (UPR) process, during L. pneumophila infection. Ectopically expressed LegU1 localizes to the endoplasmic reticulum (ER) through the region from the 88th to the 136th residue. Deletion of LegU1 increases the protein level of BiP during L. pneumophila infection. We finally indicate that LegU1 interacts with and mediates the ubiquitinational degradation of BiP. Altogether, our study identifies BiP as a new substrate of LegU1 and provides new insight into how L. pneumophila modulates the host UPR pathway during infection.

To enable adeno-associated viral vectors (AAV) to achieve their maximum potential, next-generation manufacturing processes must be developed to make gene therapies more affordable and accessible. This study focused on the design of two different intensified AAV downstream manufacturing processes at bench and pilot scale. Novel clarification methods were studied at bench scale, including the use of BioOptimal™ MF-SL tangential flow microfilters for continuous removal of cell debris. Membrane adsorbers were used for further clarification, including DNA removal. Single pass tangential flow filtration (SPTFF) was implemented at bench scale by feeding the clarified cell lysate (CCL) into two Pellicon XL50 cassettes with 100 kDa regenerated cellulose membranes. At pilot scale, a multi-membrane staged SPTFF module was designed to concentrate 10 L of AAV CCL. Both SPTFF systems provided 12X inline volumetric concentration with AAV yield > 99% after an appropriate buffer chase. Host cell protein removal was 48% and 37% for the bench and pilot scale processes, respectively. As an initial proof-of-concept, an integrated process was developed at pilot-scale which linked clarification, SPTFF, and affinity chromatography. The integrated process offered an 81% reduction in total operating time (due to the reduced volume of load material for the affinity column after preconcentration by SPTFF), 36% improvement in affinity resin utilization (due to the higher AAV concentration in the column load), and an estimated 10% reduction in raw material costs. These improvements translated to an 8.5-fold increase in overall productivity compared to an equivalent batch process, underscoring the potential for SPTFF to intensify large-scale AAV downstream processing.

Comparison of anion-exchange chromatography matrices for purification of linear and supercoiled plasmid in a direct lysate workflow.

Suspension MDCK cells are a substrate for producing influenza A virus (IAV) and typically show very high virus yields compared to other animal cells. Due to the significant heterogeneity within cell populations, studying and comparing clonal cell lines with regard to specific properties, such as superior growth or higher productivity, could facilitate process optimization. In this study, we analyzed the expressed proteins of two clonal cell lines to identify intrinsic characteristics of effective IAV producers. We compared proteome changes in two human IAV PR8 (H1N1, A/PR/8/34) infected monoclonal suspension MDCK cell lines: C59, a low-yield IAV producer with fast cell growth and small cell diameter, and C113, a high-yield IAV producer with average cell growth and large cell diameter. We examined growth rate, size, metabolism and IAV production. A total of 5177 host cell proteins were detected in both cell lines using DIA-PASEF mode with a TimsTOFpro mass spectrometer. Analysis of the differentially expressed proteins revealed that fatty acid oxidation and branched-chain amino acid degradation were upregulated in highly productive cells. In contrast, steroid biosynthesis and DNA replication were more active in faster-growing cells. Following infection, 122 proteins were significantly upregulated (p < 0.05, log2-fold change ≥1) in the high-producing cell line. These proteins were associated with membrane trafficking, interactions with the IAV-NS1 protein and virus production. Additionally, 98 proteins associated with antiviral pathways such as the proto-oncogenic receptor tyrosine kinase MET and tumor necrosis factor (TNF) signaling were downregulated (p < 0.05, log2-fold change ≤1). In the cell line that produced lower IAV PR8 titers, 77 proteins were downregulated and 57 were upregulated after infection. RNA metabolism appeared to be downregulated, while the tricarboxylic acid (TCA) cycle and the stress response were both upregulated. In the high-yield C113 clone, only proteins associated with apoptosis and the target of rapamycin kinase (TOR) were expressed following infection. This may indicate a more effective release of virus particles. A comparison of intracellular IAV PR8 protein levels demonstrated that M1 and NA levels were 4-fold and 8-fold higher, respectively, for the high-yield C113 cell line. These findings again suggest an improved virus release.

The RIG-I-like receptor family of immune proteins.

Respiratory syncytial virus (RSV) is one of the leading causes of infant hospitalization and mortality worldwide. RSV pathogenesis is a result of various virus-host interactions. While significant work has been done to elucidate mechanisms of RSV pathogenesis at a systemic level from the host perspective, here we examine how RSV pathogenesis occurs on a molecular level. While each RSV protein plays an essential role in establishing and advancing disease, each one also executes multifaceted strategies for evasion of host detection. In this review, we outline how each component of the RSV replication cycle works to co-opt host cell proteins and modulate host immune responses during entry, transcription, replication, translation, assembly, and egress. We examine the latest literature regarding RSV protein function and discuss outstanding questions in the field.

High throughput process development (HTPD) has been widely adopted for efficient development and optimization of chromatographic operations in monoclonal antibody (mAb) purification. However, the integration of non-chromatographic unit operations, particularly depth filtration following protein A chromatography, which is essential for the removal of process- and product-related impurities prior to the ion exchange chromatography (IEX) operations, remains a challenge due to the absence of commercially available micro-scale depth filtration tools. This limits the integration of this unit operation within the purification sequence, restricting the analysis of process interactions and overall process understanding. In this study, a micro-scale HTPD platform was designed and evaluated to enable integration of a depth filtration mimic, Sartobind® Q anion exchange adsorber, within a mAb purification sequence. This was achieved by translating laboratory-scale protocols to the micro-scale using workflow design tools and executed on an automated liquid handling system. Step yields and impurity clearance were assessed to confirm the equivalence of scale-down. The Sartobind® Q membrane achieved effective removal of host cell DNA (hcDNA), while subsequent IEX operations removed host cell proteins (HCPs) and high molecular weight components (HMWC), meeting target product quality specifications. The platform demonstrated robustness across varying impurity profiles, supporting its applicability for diverse process intermediates. Comparative analysis with laboratory-scale operations confirmed the performance and scalability of the micro-scale system, reducing the total run time by greater than 50%. The integrated HTPD platform offers a resource-efficient, scalable approach for comprehensive mAb purification process development and is suitable for developability assessments during early-stage development.

Polysorbates (PS), particularly polysorbate 20 (PS20) and polysorbate 80 (PS80), are widely utilized as non-ionic surfactants in biopharmaceutical formulations. Their primary role is to stabilize therapeutic proteins against interfacial stresses encountered during manufacturing, handling, and storage. Despite their widespread use, PS are complex and heterogeneous mixtures prone to degradation. The two primary degradation pathways are hydrolysis and oxidation. Hydrolytic degradation is often enzyme-mediated, commonly by residual host cell proteins such as lipases and phospholipases. Oxidative degradation can be initiated by light, temperature, metal ions, or peroxides present as impurities in raw materials. PS degradation yields various products, including free fatty acids (FFA), short-chain organic acids, aldehydes, ketones, and peroxides. The complexity of PS and their degradation products necessitates the use of sophisticated analytical methods for comprehensive characterization and monitoring. Techniques such as liquid chromatography coupled with mass spectrometry (LC-MS), charged aerosol detection (CAD), or evaporative light scattering detection (ELSD), as well as specific assays for FFA and peroxides, are employed. A significant concern arising from PS degradation is the formation of visible and subvisible particles, often composed of poorly soluble FFAs. While PS degradation and particle formation may not always impact protein quality attributes such as aggregation or biological activity under certain conditions, their potential consequences on product quality, safety, and compliance require careful consideration. Effective mitigation and control strategies involve stringent raw material qualification, optimization of manufacturing processes, robust formulation development, and comprehensive stability testing with appropriate analytical methods.

Integrated native digestion and molecular weight cutoff enrichment with DIA-MS: a sensitive and selective approach for quantitative analysis of HCP.

Hydrophobicity analysis of Chinese Hamster Ovary host cell proteins using analytical hydrophobic interaction chromatography and LC-MS.

Composition, three-dimensional structure and formation mechanism of the foot and mouth disease virus replication complexes.

PAT-on-a-chip: Miniaturization of analytical assays towards data-driven bioprocess development and optimization.

Image based subcellular mapping of the protein landscape of SARS-CoV-2 infected cells for target-centric drug repurposing.

Mitigating polysorbate degradation-induced visible particle formation in an antibody formulation: From root cause identification to strategic solution.

Minute virus of canines (MVC), which is a member of the Bocaparvovirus genus, is a non-enveloped, single-stranded DNA virus that causes respiratory and gastrointestinal disease in canines, as well as causing infertility and fetal death in pregnant dogs. The non-structural small protein NP1 of bocaparvoviruses is a unique feature that distinguishes the bocaparvovirus subfamily from other parvovirus subfamilies. In the life cycle of the MVC, NP1 plays an indispensable role in viral DNA replication and pre-mRNA processing. Currently, there is a paucity of studies reporting the characterization of host cell proteins interacting with NP1 during MVC replication. In this study, we screened and identified host cell proteins interacting with MVC-NP1 through immunoprecipitation (IP) combined with liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis; MCM7 (Mini-chromosome Maintenance Protein 7) has been identified and confirmed to interact directly with NP1 through its N-terminal domain. Furthermore, functional studies reveal that MCM7 is essential in MVC replication. The knockdown of MCM7 decreased the expression of this MVC protein significantly, as well as suppressing MVC replication by arresting the cell cycle in the G0/G1 phase during infection. Conversely, up-regulating MCM7 can rehabilitate the expression of MVC proteins, as well as supporting MVC replication. In conclusion, this study elucidates the interaction between the NP1 protein of MVC and the host factor MCM7, demonstrating that MCM7 is a key factor in the replication process of MVC. These findings provide a potential target for future antiviral therapy.

Efficient host cell protein clearance: A study of membrane adsorbers and resins in biopharmaceutical processes.