Effects of ionic strength on lysozyme uptake rates in cation exchangers. I: Uptake in SP Sepharose FF

Abstract

Fluorescence scanning confocal microscopy was used in parallel with batch uptake and breakthrough measurements of transport rates to study the effect of ionic strength on the uptake of lysozyme into SP Sepharose FF. In all cases the adsorption isotherms were near-rectangular. As described previously, the intraparticle profiles changed from slow-moving self-sharpening fronts at low salt concentration, to fast-moving diffuse profiles at high salt concentration, and batch uptake rates correspondingly increased with increasing salt concentration. Shrinking core and homogeneous diffusion frameworks were used successfully to obtain effective diffusivities for the low salt and high salt conditions, respectively. The prediction of column breakthrough was generally good using these frameworks, except for low-salt uptake results. In those cases, the compressibility of the stationary phase coupled with the shrinking core behavior appears to reduce the mass transfer rates at particle-particle contacts, leading to shallower breakthrough curves. In contrast, the fast uptake rates at high ionic strength appear to reduce the importance of mass transfer limitations at the particle contacts, but the confocal results do show a flow rate dependence on the uptake profiles, suggesting that external mass transfer becomes more limiting at high ionic strength. These results show that the complexity of behavior observable at the microscopic scale is directly manifested at the column scale and provides a phenomenological basis to interpret and predict column breakthrough. In addition, the results provide heuristics for the optimization of chromatographic conditions.

Impact of denaturation with urea on recombinant apolipoprotein A‐IMilano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

“…(4) and (5) predict a shock transition in the adsorbed phase protein concentration or "shrinking core" behavior. This phenomenon has been observed at the microscopic level for Sepharose FF cation-exchangers using fluorescently labeled proteins [30,31]. Eqs.…”

“…For bioprocess applications where large diameter adsorbent beads and high mobile phase velocities are typically utilized, chromatographic efficiency is nearly completely determined by mass transfer kinetics [25]. Accordingly, numerous studies have been devoted to protein mass transfer in chromatography media (e.g., [26][27][28][29][30][31]). However, to our knowledge, the effects of urea on protein-binding capacity, chromatography retention, and intraparticle mass transfer in ion-exchange columns have not been investigated in a systemic fashion.…”

Chromatography of proteins on charge-variant ion exchangers and implications for optimizing protein uptake rates

“…This is done by capturing the intraparticle uptake profiles and fitting the observed concentration gradients to a representative model, with most attention being paid to the association of a sharp or a diffuse front to the pore diffusion or the homogeneous diffusion model respectively [18][19][20][21][22][23][24][25]. This method has been used to explore the roles of solution conditions, adsorbent structure and protein structure in determining the transport mechanism.…”

“…This method has been used to explore the roles of solution conditions, adsorbent structure and protein structure in determining the transport mechanism. Specifically, in some situations the uptake mechanism has been observed to change with solution conditions, primarily an increase in ionic strength [18][19][20][21][22]26]. The change in uptake mechanism was accompanied by an enhancement in the overall transport rate, as reflected in higher effective diffusivities in batch uptake experiments [19].…”

“…The change in uptake mechanism appears to have important implications for process chromatography as breakthrough profiles at higher flow rates are significantly sharper at ionic strengths at which the homogeneous diffusion model applies [18,20]. Harinarayan et al [26] found similar behavior in identifying maxima in the dynamic binding capacities of monoclonal antibodies (mAbs) as a function of conductivity (hence ionic strength).…”