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Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra-scale down shearing device.

Hassan S, Keshavarz-Moore E, Ma J, Thomas C - Biotechnol. Bioeng. (2013)

Bottom Line: A possible method for extraction of MAbs from roots is by homogenization, breaking the roots into fragments to release the antibody.Size distributions of the remaining intact roots and root fragments were obtained as a function of shearing time.It was postulated that root breakage in such a high shearing device was due to root-impeller collisions and the particle size data suggest that roots colliding with the impeller were completely fragmented into debris particles of the order of 0.1 mm in length.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, London, United Kingdom.

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Methodology for root debris size analysis. Light microscopic images were taken at 2.5× magnification for individual samples at each time point. Image J software was used to analyse particle sizes. a–e represents the flow diagram of the image processing algorithm for the analysis. a: A raw image of root debris post-shearing for 240 s, converted to greyscale. b: A thresholded binary image with the particles in white against a black background. c: Using the Freemanual selection tool, the area for analysis has been selected, clearing any shading (here the top right hand corner of the image) caused by shadows of the microscope lens' outer edge, and the image inverted. The particles appear black against a white background. d: Apparent holes or gaps within the root pieces have been filled using the Binary option of the software. e: Using a measured calibration factor of 144 pixels mm−1, only root particles of projected area greater than 0.01 mm2 were measured, assuming that anything less than this was likely to be adventitious debris and dust, and the feret function (defined by the software as the longest distance between any two points along the selection boundary), considered to be the fragment length.
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fig03: Methodology for root debris size analysis. Light microscopic images were taken at 2.5× magnification for individual samples at each time point. Image J software was used to analyse particle sizes. a–e represents the flow diagram of the image processing algorithm for the analysis. a: A raw image of root debris post-shearing for 240 s, converted to greyscale. b: A thresholded binary image with the particles in white against a black background. c: Using the Freemanual selection tool, the area for analysis has been selected, clearing any shading (here the top right hand corner of the image) caused by shadows of the microscope lens' outer edge, and the image inverted. The particles appear black against a white background. d: Apparent holes or gaps within the root pieces have been filled using the Binary option of the software. e: Using a measured calibration factor of 144 pixels mm−1, only root particles of projected area greater than 0.01 mm2 were measured, assuming that anything less than this was likely to be adventitious debris and dust, and the feret function (defined by the software as the longest distance between any two points along the selection boundary), considered to be the fragment length.

Mentions: Approximately 0.35 g of 10 mm roots were sheared in 7 mL of 1× PBS buffer at 75 s−1 for 30, 120, 240, or 360 s. At the end of each shearing time, and a control where no shearing was performed (i.e., 0 s), the resulting debris and liquid was centrifuged at 4,000 rpm, for 20 min at 4°C. Shearing experiments were performed in triplicate. The remaining intact roots, that is, those that were still 10 mm in length were blotted dry to remove excess surrounding liquid and weighed. The number of remaining intact roots was calculated as their total mass divided by the average mass of a single intact root (0.00281 ± 0.0002 g). The subsequent estimation of the fraction of remaining intact roots after shear as the final mass of intact roots over the initial root mass (∼0.35 g) was used in Equation 1. To analyze the distribution of the remaining fragments, three images of a 0.1 mL sample of the fragments were imaged using a LEICA DM light microscope (Leica Microsystems Ltd, Heerbrugg, Switzerland) at a magnification of 2.5 times, and root debris lengths characterized using Image J software (Rasband and Bright, 1995). Details of how the software was used to measure fragment length is explained in the legend of Figure 3. This analysis was done in triplicate and the measurements combined to give the fragment length distributions. The frequency of fragments at each length was determined by counting the number of fragments of each length, using the “COUNTIF” function in Excel 2007. Since the sampled images represented a total of 0.3 mL of the total sample in the shearing device, the numbers of each fragment length was multiplied by 20 for an approximate total number in the 6 mL sample (7 mL total volume but 1 mL removed immediately after shearing and centrifugation for IgG analysis).


Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra-scale down shearing device.

Hassan S, Keshavarz-Moore E, Ma J, Thomas C - Biotechnol. Bioeng. (2013)

Methodology for root debris size analysis. Light microscopic images were taken at 2.5× magnification for individual samples at each time point. Image J software was used to analyse particle sizes. a–e represents the flow diagram of the image processing algorithm for the analysis. a: A raw image of root debris post-shearing for 240 s, converted to greyscale. b: A thresholded binary image with the particles in white against a black background. c: Using the Freemanual selection tool, the area for analysis has been selected, clearing any shading (here the top right hand corner of the image) caused by shadows of the microscope lens' outer edge, and the image inverted. The particles appear black against a white background. d: Apparent holes or gaps within the root pieces have been filled using the Binary option of the software. e: Using a measured calibration factor of 144 pixels mm−1, only root particles of projected area greater than 0.01 mm2 were measured, assuming that anything less than this was likely to be adventitious debris and dust, and the feret function (defined by the software as the longest distance between any two points along the selection boundary), considered to be the fragment length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352334&req=5

fig03: Methodology for root debris size analysis. Light microscopic images were taken at 2.5× magnification for individual samples at each time point. Image J software was used to analyse particle sizes. a–e represents the flow diagram of the image processing algorithm for the analysis. a: A raw image of root debris post-shearing for 240 s, converted to greyscale. b: A thresholded binary image with the particles in white against a black background. c: Using the Freemanual selection tool, the area for analysis has been selected, clearing any shading (here the top right hand corner of the image) caused by shadows of the microscope lens' outer edge, and the image inverted. The particles appear black against a white background. d: Apparent holes or gaps within the root pieces have been filled using the Binary option of the software. e: Using a measured calibration factor of 144 pixels mm−1, only root particles of projected area greater than 0.01 mm2 were measured, assuming that anything less than this was likely to be adventitious debris and dust, and the feret function (defined by the software as the longest distance between any two points along the selection boundary), considered to be the fragment length.
Mentions: Approximately 0.35 g of 10 mm roots were sheared in 7 mL of 1× PBS buffer at 75 s−1 for 30, 120, 240, or 360 s. At the end of each shearing time, and a control where no shearing was performed (i.e., 0 s), the resulting debris and liquid was centrifuged at 4,000 rpm, for 20 min at 4°C. Shearing experiments were performed in triplicate. The remaining intact roots, that is, those that were still 10 mm in length were blotted dry to remove excess surrounding liquid and weighed. The number of remaining intact roots was calculated as their total mass divided by the average mass of a single intact root (0.00281 ± 0.0002 g). The subsequent estimation of the fraction of remaining intact roots after shear as the final mass of intact roots over the initial root mass (∼0.35 g) was used in Equation 1. To analyze the distribution of the remaining fragments, three images of a 0.1 mL sample of the fragments were imaged using a LEICA DM light microscope (Leica Microsystems Ltd, Heerbrugg, Switzerland) at a magnification of 2.5 times, and root debris lengths characterized using Image J software (Rasband and Bright, 1995). Details of how the software was used to measure fragment length is explained in the legend of Figure 3. This analysis was done in triplicate and the measurements combined to give the fragment length distributions. The frequency of fragments at each length was determined by counting the number of fragments of each length, using the “COUNTIF” function in Excel 2007. Since the sampled images represented a total of 0.3 mL of the total sample in the shearing device, the numbers of each fragment length was multiplied by 20 for an approximate total number in the 6 mL sample (7 mL total volume but 1 mL removed immediately after shearing and centrifugation for IgG analysis).

Bottom Line: A possible method for extraction of MAbs from roots is by homogenization, breaking the roots into fragments to release the antibody.Size distributions of the remaining intact roots and root fragments were obtained as a function of shearing time.It was postulated that root breakage in such a high shearing device was due to root-impeller collisions and the particle size data suggest that roots colliding with the impeller were completely fragmented into debris particles of the order of 0.1 mm in length.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, University College London, London, United Kingdom.

Show MeSH
Related in: MedlinePlus