Fractal dimension of antibody‐PEG precipitate: Light microscopy for the reconstruction of 3D precipitate structures

Abstract

Protein and in particular antibody precipitation by PEG is a cost‐effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo‐TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.1–0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro‐ to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.

Characterization of hydrodynamics and volumetric power input in microtiter plates for the scale‐up of downstream operations

“…The lower shear stress in the 96‐well plates compared with the 6‐well plates has already been shown via the clay floc destruction method used in our previous research (Dürauer et al, 2016 ) as well as by Wutz et al ( 2018 ). Recent results also confirm the impact of the shaking diameter on the shear rate, which could be limiting for choosing the operation conditions for the development of different downstream processes such as the conditions for the precipitation of antibodies, since it can compromise the particle size distribution of the precipitates (Satzer et al, 2020 ). Thus, for those unit operations, the decision of the system cannot only rely on the conservation of volumetric power input.…”

“…Process engineers are studying strategies to implement affordable unit operations while maintaining high product yields and purity. One operation of choice is typically precipitation, which is very efficient for the primary recovery of antibodies (Hammerschmidt et al, 2016 ; Satzer et al, 2020 ). However, the efficient scale‐up of such unit operations is not straightforward.…”

Design of millidevices to expedite apparent solubility measurements

“…These results again show that the variation could be produced by the shear stress inducing different particle sizes and size distributions. 50,53…”

“…These results again show that the variation could be produced by the shear stress inducing different particle sizes and size distributions. 50,53 Double-strand DNA precipitation with CaCl 2 . Impurities present in the cell culture supernatants such as HCP and dsDNA can be easily precipitated by pH precipitation 54 and precipitating agents like CaCl 2 8,55 or CA.…”

Intensified capture of recombinant blood coagulation factors by continuous, integrated downstream processing

“…We think that these differences are due to the operation mode itself. Batch precipitation typically results in a different floc size distribution as compared to continuous precipitation or stepwise addition 30,31 . In batch mode, a single addition of precipitant leads to a certain number of nuclei and they will continue to grow.…”