Recent Breakthroughs

Smith J, Richardson A, Beaumal C, Ainciburu M, Carillo S, Pashkova A, Arrey TN, Damoc NE, Clarke C, Bones J.

J Am Soc Mass Spectrom

PMID:41996158

Free PMC article

Host cell proteins (HCPs) are endogenous proteins generated in cellular production systems alongside the biotherapeutic of interest. Removal of HCPs is crucial as they can be detrimental to product efficacy and patient safety. Due to its ability to determine individual HCP concentrations, liquid chromatography tandem mass spectrometry is increasingly utilized as an orthogonal method to ELISA for HCP monitoring. For protein biotherapeutics like monoclonal antibodies, their dynamic range makes detection of low-level HCPs difficult. The Orbitrap Astral MS has the potential to overcome such challenges, offering improvements in protein identifications in complex sample matrices while simultaneously reducing analysis times. Here, we utilize the Orbitrap Astral MS to perform HCP analysis on 36 protein biotherapeutics. Our workflow used a short 60 samples-per-day separation method and was initially benchmarked against four previously published studies, demonstrating comparable levels of HCP identifications. 236 HCPs were detected across the cohort and 55% of those found to be quantifiable in at least one product using label free quantitation. Functional analysis revealed that most detected HCPs had functions related to catalysis or binding, predominately catalytic activity (46%, 97 gene IDs) or protein binding (44%, 91 gene IDs). Nearly 80% of quantifiable HCPs were detected at concentrations below 10 ng/mg, with 8% detected below concentrations of 1 ng/mg. These included HCPs considered as "high-risk" by the Biophorum Development Group. This study shows how new generation mass spectrometry instruments can enable detection of low-level HCPs while allowing for a rapid and more informed understanding of a product's HCP content.

Montasser AA, Mohamed SNA, Ali AAB.

Sci Rep

PMID:41998046

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Hyalomma dromedarii is a hard tick species parasitizing domestic animals, particularly camels. Heavy infestation results in huge economic losses through severe blood loss and transmission of pathogens, in addition to crucial problems for camel production. Worldwide control of ticks is mainly based on acaricides, which have led to environmental pollution, resistance development, and an increase in the cost of control. To reduce the drawbacks of chemical acaricides, new tick control methods are therefore required, such as the application of natural plant extracts. Citrullus colocynthis, commonly known as bitter apple, is a desert plant found in Egypt. It has an economic importance due to its bioactive compounds with antidiabetic, antimicrobial, and potentially anticancer properties. In addition, it is used as a natural preservative, as it was historically applied to protect Egyptian manuscripts and leather from fungal damage. The goal of this work was to study the histopathological and ultrastructural changes of H. dromedarii integument after immersion in 100 mg/mL of C. colocynthis ethanolic extract. Volatile components of the extract were detected following the use of gas chromatography-mass spectrometry (GC-MS). Light, scanning, and transmission electron microscopy examinations provided evidence that C. colocynthis caused great damage to the integument. Increasing eroded areas with irregular folds and warts were observed by SEM. LM and TEM showed integumental layers separation, procuticle disorganization, subcuticular layer rupture and epidermal layer damage. GC-MS revealed volatile constituents, such as methyl linoleate, octadecadienoic, palmitic, and stearic acids. This is the first histological investigation that reported the integumentary damage caused by C. colocynthis in H. dromedarii. The present data suggest that the changes in all integument layers of the female tick H. dromedarii following treatment with C. colocynthis extract may facilitate the transport of toxic compounds into ticks' internal systems, which can then affect other organs. As a result, C. colocynthis can be considered as a promising tick control agent.

Leibiger TM, Min L, Lee KH.

Hum Gene Ther

PMID:41992847

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Scalable purification platforms have been developed for adeno-associated virus (AAV) processing to support large-scale vector manufacturing. The ability of column chromatography to recover packaged vectors and remove empty capsids has been well-established, but knowledge gaps remain for understanding process parameter impacts on impurity retention. In this work, we examine the impacts of two key process parameters-the harvest method and affinity resin selection-on residual host cell protein (HCP) retention. Sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics is applied to comprehensively profile residual HCPs in affinity chromatography (AC) elution pools from four AAV serotypes (AAV2, -5, -8, and -9) produced by suspension HEK293 cells. Vectors were purified from cell culture lysates and from supernatants using POROS ™ CaptureSelect ™ AAVX (AAVX) and one additional serotype-specific affinity resin-Capto ™ AVB (AVB), POROS CaptureSelect AAV8 (PAAV8), or POROS CaptureSelect AAV9 (PAAV9). Significant divergence in residual HCP profiles was observed with the use of different affinity resins, with AVB and PAAV9 showing reduced residual HCP content in elution pools compared with AAVX and PAAV8. Processing of null culture lysates with fresh resins and resins with digested single-domain antibody fragments (sdAbs) shows that differences in resin performance are driven by variable nonspecific sdAb association with cellular impurities. Proteomic analysis of vector preparations from lysates compared with supernatants demonstrates product quality advantages of designing a media-only harvest process, specifically for AAV8, which was measured to contain an average of 66% of total vector genome content in the cell culture media. This work highlights the importance of serotype-specific tailoring of AAV downstream process design for improved product quality attributes to support clinical manufacture and small-scale analytics workflows.

Cao Y, Chen W, Li Q, Liao Y, Wang J, Pan L.

J Agric Food Chem

PMID:41995085

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Plant-microbiome interactions are essential for plant health and productivity under stress; however, little is known about these interactions in response to herbicide. Here, we integrated 16S rRNA gene sequencing with nontargeted gas chromatography-mass spectrometry (GC-MS) to investigate the interactions between rhizosphere microbiomes and metabolomes in Polypogon fugax . The results indicated that quizalofop- p -ethyl-resistant (QU-resistant) P. fugax promoted microbial colonization within its microbiome, enriched the abundance of Verrucomicrobia, and increased the levels of d-proline and α,α-trehalose in the rhizosphere, potentially attracting Verrucomicrobia. Furthermore, when the rhizosphere microbiome from R3 populations was transplanted to QU-sensitive plants, the recipients exhibited enhanced antioxidant defense systems and demonstrated reduced sensitivity to QU. These results suggest that the rhizosphere microbiome of QU-resistant P. fugax contributes to its resistance against QU. Overall, our findings highlight the complex interactions among herbicide resistance mechanisms, rhizosphere microbiota, and plant responses, suggesting potential strategies for managing herbicide-resistant weed populations.

de Sá HC, Rodrigues GM, de Carvalho VMP, Silva LP, Kato MJ, Estrela-Lima A, Canuto GAB.

Metabolomics

PMID:41998452

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Background and aims The development of breast cancer exhibits a heterogeneous and complex character in both felines and humans, which motivates the search for serum biomarkers for diagnosis, prognosis, and therapeutic monitoring. Although the feline species is recognized as a relevant comparative model for human breast cancer, metabolomic studies in cats are still scarce. This work aimed to investigate altered serum metabolites involved in feline mammary carcinoma (FMC). Methods Serum samples from 28 adult female cats (11 healthy and 17 with malignant mammary tumors undergoing mastectomy) were evaluated. The samples were extracted with pure methanol and derivatized (oximation followed by silylation) for GC-MS analysis. Results Twenty-six metabolites or chemical classes were found significantly altered. The main alteration was related to the metabolism of amino acids, carbohydrates, and the tricarboxylic acid cycle, mostly with reduced serum levels in female cancer patients, suggesting high tumor uptake. Analysis of metabolic pathways revealed alterations in the metabolism of alanine, aspartate, and glutamate; arginine and proline; starch and sucrose; and butanoate metabolism. Conclusions These findings provide preliminary evidence supporting the determination of candidate biomarkers and the mapping of disrupted metabolic pathways in FMC. Validation of the study and confirmation of the metabolite's structural identity are critical to enable robust comparative studies and to direct the development of innovative diagnostic and therapeutic strategies.