Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

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A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse

Molecules ◽

10.3390/molecules26144203 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4203

Author(s):

Héloïse Débare ◽

Nathalie Moiré ◽

Firmin Baron ◽

Louis Lantier ◽

Bruno Héraut ◽

...

Keyword(s):

Protein Kinase ◽

Toxoplasma Gondii ◽

Intraperitoneal Administration ◽

Congenital Toxoplasmosis ◽

Parasite Burden ◽

Oral Route ◽

Dependent Protein Kinase ◽

Calcium Dependent ◽

Dependent Protein ◽

Calcium Dependent Protein Kinase

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

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Thyrotropin-releasing hormone rapidly and transiently stimulates cytosolic calcium-dependent protein phosphorylation in GH3 pituitary cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)34417-x ◽

1982 ◽

Vol 257 (13) ◽

pp. 7566-7573 ◽

Cited By ~ 15

Author(s):

D S Drust ◽

T F Martin

Keyword(s):

Protein Phosphorylation ◽

Cytosolic Calcium ◽

Thyrotropin Releasing Hormone ◽

Pituitary Cells ◽

Releasing Hormone ◽

Calcium Dependent ◽

Dependent Protein

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Genetic mapping and transcriptomic characterization of a new fuzzless-tufted cottonseed mutant

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa042 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Qian-Hao Zhu ◽

Warwick Stiller ◽

Philippe Moncuquet ◽

Stuart Gordon ◽

Yuman Yuan ◽

...

Keyword(s):

Molecular Mechanisms ◽

Agronomic Traits ◽

Gene Families ◽

Mutant Phenotype ◽

Wild Type ◽

Calcium Dependent ◽

Dependent Protein ◽

Flanking Region ◽

Comparative Transcriptomic Analysis

Abstract Fiber mutants are unique and valuable resources for understanding the genetic and molecular mechanisms controlling initiation and development of cotton fibers that are extremely elongated single epidermal cells protruding from the seed coat of cottonseeds. In this study, we reported a new fuzzless-tufted cotton mutant (Gossypium hirsutum) and showed that fuzzless-tufted near-isogenic lines (NILs) had similar agronomic traits and a higher ginning efficiency compared to their recurrent parents with normal fuzzy seeds. Genetic analysis revealed that the mutant phenotype is determined by a single incomplete dominant locus, designated N5. The mutation was fine mapped to an approximately 250-kb interval containing 33 annotated genes using a combination of bulked segregant sequencing, SNP chip genotyping, and fine mapping. Comparative transcriptomic analysis using 0–6 days post-anthesis (dpa) ovules from NILs segregating for the phenotypes of fuzzless-tufted (mutant) and normal fuzzy cottonseeds (wild-type) uncovered candidate genes responsible for the mutant phenotype. It also revealed that the flanking region of the N5 locus is enriched with differentially expressed genes (DEGs) between the mutant and wild-type. Several of those DEGs are members of the gene families with demonstrated roles in cell initiation and elongation, such as calcium-dependent protein kinase and expansin. The transcriptome landscape of the mutant was significantly reprogrammed in the 6 dpa ovules and, to a less extent, in the 0 dpa ovules, but not in the 2 and 4 dpa ovules. At both 0 and 6 dpa, the reprogrammed mutant transcriptome was mainly associated with cell wall modifications and transmembrane transportation, while transcription factor activity was significantly altered in the 6 dpa mutant ovules. These results imply a similar molecular basis for initiation of lint and fuzz fibers despite certain differences.

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