Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy

Abstract

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.

Evaluation of the impact of lipid fouling during the chromatographic purification of virus‐like particles from Saccharomyces cerevisiae

“…8 -10 The majority of studies have focused on the clogging of solids within the column, which results in uneven distribution and channelling of the flow. 11 Findings on how fouling affects the capacity and breakthrough characteristics in ion exchange chromatography (IEX), 7 and a later study 12 to visualize matrix fouling on individual beads using confocal scanning laser microscopy have laid down a solid foundation in terms of utilizing these techniques to study other process feeds and matrices. However, these previous studies focused on protein and nucleic acid fouling from an artificial and hence vastly simplified feed stream.…”

Confocal laser scanning microscopy as an analytical tool in chromatographic research

“…Commercially available molecules or in-house conjugates with higher DoL values might thus lead to clear deviations between native and labeled molecules. In contrast to this, Siu et al [20], for example, claim that in there system (BSA on Q Sepharose FF) no significant changes for bio-conjugates with a DoL of approximately 3 using Cy5.5 (net charge -3) as fluorescent dye could be found, even though this combination results in a charge shift of approximately -12 (see Table 2). …”

“…The approach followed by Siu et al in their study on fouling of chromatographic resins to re-equilibrate the respective resins into one buffer system prior to CLSM analysis might solve the issue of different fluorescent intensities. However, any change in buffer system may alter the chromatographic system-the significance of this change depends on the respective system [20].…”

“…This study further underlines the restricted use of non-diluted commercially available protein-dye conjugates. Next to the fluorescence detected from the adsorbed protein-dye conjugates, some adsorbent materials will impair CLSM analysis due to their auto-fluorescence at a certain wavelength [20,25]. Only when using the correct filters for the CLSM settings, this auto-fluorescence could be separated from the fluorescence intensities obtained by the protein-dye conjugates.…”

Impact of clarification strategy on chromatographic separations: Pre‐processing of cell homogenates

“…Visualization of the data in this manner shows a cumulative reduction in HIC column capacity with increasing levels of lipid and debris, it can be seen capacity is nearly halved in the worst conditions studied. The mechanism by which debris reduces HIC capacity is via binding to the surface of beads as has been reported previously (Siu et al, 2006a). The action of lipids is less clear, they likely bind within the porous structure of the bead.…”