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Exploring the Invisible: TEM expertise for powerful sample analysis

Negative staining: Clarity in shadows

For the detection of adventitious agents in bulk harvest samples and the qualitative and quantitative analysis of suspended particles in liquid sample matrices, we utilise the method of negative staining TEM (nsTEM).

The test item is embedded in a heavy metal salt solution which acts as a contrasting agent deflecting electrons due to its high electron density. As a result the embedded particles appear bright against a dark background when imaged using transmission electron microscopy.

nsTEM is also ideally suited to gain visual feedback on your sample quality in the various steps of upstream and downstream processing (e.g. detect background particles, cell debris, sample impurities, aggregates).

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Our method of nsTEM for the detection of adventitious agents in unprocessed bulk harvest samples

Retrovirus-like particles which are C-type particles are released directly into the cell culture media and should be detected. Furthermore, additional adventitious agents which could have been introduced to the process should also be detected. Unprocessed cell-free culture supernatant is thus investigated using negative staining TEM

After separating the cells from the test item via centrifugation, possible virus or virus-like particles can be concentrated in the supernatant by ultracentrifugation. A defined volume of this test item is then mixed with reference beads and stained with heavy metal salts. Biological structures are embedded in the staining agent and not directly stained (negative staining). These structures then appear bright against a dark background when visualising with TEM.

To ensure that the test is executed in the defined parameters of the validation, system suitability criteria (SSC) were defined. Only after all SSCs have been successfully passed, the test item grid is then screened for adventitious agents.

If virus or virus-like particles are detected, they are counted and characterised. The concentration of the introduced reference beads is known and the ratio of reference beads to virus or virus-like particles is used to calculate the quantity of the respective particles.

Results and the corresponding Final Reports (CoA) are issued within 15 working days.