High throughput inclusion body sizing: Nano particle tracking analysis. Issue 6 (28th April 2017)

• Record Type: Journal Article
• Authors: Reichelt, Wieland N.; Kaineder, Andreas; Brillmann, Markus; Neutsch, Lukas; Taschauer, Alexander; Lohninger, Hans; Herwig, Christoph
• Is Part Of: Biotechnology journal. Volume 12:Issue 6(2017)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-04-28
• Subjects: Electron microscopy -- Grey scale image segmentation -- Inclusion body -- Nano particle tracking analysis -- Stickiness; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201600471
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle‐based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non‐fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non‐fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle‐based, fast and high throughput method (1000–3000 particles), thus constituting a much more accurate and representative analysis than currently available methods. Abstract : To investigate the interlink of upstream and downstream processing a single particle based analysis of the end product of upstream processing – the inclusion bodies (IBs) – is required. In this context, Nano particle tracking analysis (NTA) offers an approach to size a large number of IBs in a comparably short amount of time. Using transmission electron microscopy in combination with manual grey scale image segmentation facilitated a relative verification of the NTA results. Besides establishing NTA as method for fast IB sizing, this contribution illustrates the importance of sample fixation prior to cell disruption, which suggests a so far underestimated role of high pressure homogenization in determining IB shape.