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Exosome from mouse T lymphocyte cells

ABSTRACT: Exosomes transferred from the T cell to the APC contain many different types of biologically active molecules, including proteins and genetic material. To investigate the possible specific function of the different components of the biologic material transferred from the T cell to the DC, we have characterized the protein and genetic content of EVs isolated from the culture supernatant of primary T lymphoblasts by differential ultracentrifugation

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): T Cell

SUBMITTER: Inmaculada Jorge

LAB HEAD: Francisco Sánchez-Madrid

PROVIDER: PXD008843 | Pride | 2018-05-30

REPOSITORIES: Pride

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Project description:The glycosylation of antibody-based proteins is vital in conferring the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. The simplified mass spectrometers offer ease of operation as well as the portability of method development across various operations. Furthermore, data analysis would need to have a degree of automation to relay information back to the manufacturing line. We set out to investigate the applicability of using a semiautomated data analysis workflow to investigate glycosylation in different biologic development test cases. The workflow involves data acquisition using a BioAccord LC-MS system with a data-analytical tool called GlycopeptideGraphMS along with Progenesis QI to semi-automate glycoproteomic characterisation and quantitation in complex LC-MS1 data of a glycoproteomic analysis. Data analysis which involved identifying glycopeptides and their quantitative glycosylation was performed in 30 minutes with minimal user intervention. To demonstrate the effectiveness of the antibody and biologic glycopeptide assignment in various scenarios akin to biologic development activities, we demonstrate the effectiveness in the filtering of IgG1 and IgG2 subclasses from human serum IgG as well as innovator drugs trastuzumab and adalimumab and glycoforms by virtue of their glycosylation pattern. We demonstrate a high correlation between conventional released glycan analysis with fluorescent tagging and glycopeptide assignment derived from GraphMS. GraphMS workflow was then used to monitor the glycoform of our in-house trastuzumab biosimilar produced in fed-batch cultures, and it offers valuable insights into the effect of culture media changes on bioprocess and biologic characterisation. The demonstrated utility of GraphMS to semi-automate quantitation and qualitative identification of glycopeptides proves to be an easy and scalable data analysis method that can complement emerging multi-attribute monitoring (MAM) analytical toolsets in bioprocess environments.

			Action	DRS
	exos_rep1_fr1.msf	Msf
	exos_rep1_fr1.raw	Raw
	exos_rep1_fr2.msf	Msf
	exos_rep1_fr2.raw	Raw
	exos_rep1_fr3.msf	Msf

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Exosome from mouse T lymphocyte cells

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