Physical characteristics critical to chromatography including geometric porosity and tortuosity within the packed column were analysed based upon three dimensional reconstructions of bed structure in-situ. Image acquisition was performed using two X-ray computed tomography systems, with optimisation of column imaging performed for each sample in order to produce three dimensional representations of packed beds at 3μm resolution. Two bead materials, cellulose and ceramic, were studied using the same optimisation strategy but resulted in differing parameters required for X-ray computed tomography image generation. After image reconstruction and processing into a digital three dimensional format, physical characteristics of each packed bed were analysed, including geometric porosity, tortuosity, surface area to volume ratio as well as inter-bead void diameters. Average porosities of 34.0% and 36.1% were found for ceramic and cellulose samples and average tortuosity readings at 1.40 and 1.79 respectively, with greater porosity and reduced tortuosity overall values at the centre compared to the column edges found in each case. X-ray computed tomography is demonstrated to be a viable method for three dimensional imaging of packed bed chromatography systems, enabling geometry based analysis of column axial and radial heterogeneity that is not feasible using traditional techniques for packing quality which provide an ensemble measure.

X-ray computed tomography (CT) and focused ion beam (FIB) microscopy were used to generate three dimensional representations of chromatography beads for quantitative analysis of important physical characteristics including tortuosity factor. Critical-point dried agarose, cellulose and ceramic beads were examined using both methods before digital reconstruction and geometry based analysis for comparison between techniques and materials examined. X-ray 'nano' CT attained a pixel size of 63 nm and 32 nm for respective large field of view and high resolution modes. FIB improved upon this to a 15 nm pixel size for the more rigid ceramic beads but required compromises for the softer agarose and cellulose materials, especially during physical sectioning that was not required for X-ray CT. Digital processing of raw slices was performed using software to produce 3D representations of bead geometry. Porosity, tortuosity factor, surface area to volume ratio and pore diameter were evaluated for each technique and material, with overall averaged simulated tortuosity factors of 1.36, 1.37 and 1.51 for agarose, cellulose and ceramic volumes respectively. Results were compared to existing literature values acquired using established imaging and non-imaging techniques to demonstrate the capability of tomographic approaches used here.

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

X-ray computed tomography has been demonstrated to be capable of imaging 1 mL (5 mm diameter, 50 mm height) chromatography packed beds under compression, visualising the 3D structure and measuring changes to geometry of the packing. 1 mL pre-packed columns did not exhibit any structural changes at vendor specified flow rate limits, however cellulose beds did compress at higher flow rates, which was imaged before, during and after flow. This was used to visualise and quantitate changes to porosity, tortuosity and permeability based on simulation of flow through the packed bed structure using the imaging data. When using a high flow rate it was found that a decrease in porosity could be measured during compression before reverting after flow had ceased, with corresponding changes to tortuosity and permeability also occurring. X-ray CT imaging of packed beds and individual beads exposed to foulant-rich process streams resulted in considerable image quality loss, associated with residual biological material. In order to address this, digital processing using an erosion-dilation method was applied at bead and bed scales to computationally alter the porosity by adding or removing material from the existing surface to calculate the impact upon tortuosity factor. The eroded

A multiple length scale approach to the imaging and measurement of depth filters using X-ray computed tomography is described. Three different filter grades of varying nominal retention ratings were visualized in 3D and compared quantitatively based on porosity, pore size and tortuosity. Positional based analysis within the filters revealed greater voidage and average pore sizes in the upstream quartile before reducing progressively through the filter from the center to the downstream quartile, with these results visually supported by voidage distance maps in each case. Flow simulation to display tortuous paths that flow may take through internal voidage were examined.Digital reconstructions were capable of identifying individual constituents of voidage, cellulose and perlite inside each depth filter grade, with elemental analysis on upstream and downstream surfaces confirming perlite presence. Achieving an appropriate pixel size was of particular importance when optimizing imaging conditions for all grades examined. A 3 µm pixel size was capable of representing internal macropores of each filter structure; however, for the finest grade, an improvement to a 1 µm pixel size was required in order to resolve micropores and small perlite shards. Enhancing the pixel size resulted in average porosity measurements of 70% to 80% for all grades. Graphical abstract