Roles of interstitial fraction and load conditions on the dynamic binding capacity of proteins on capillary‐channeled polymer fiber columns

Abstract

Capillary-channeled polymer (C-CP) fibers are used as a stationary phase for ion-exchange chromatography of proteins. Collinear packing of the fibers permits operation at high linear velocities (Uo > 100 mm s(-1)) and low backpressure (<2,000 psi) on analytical-scale columns. Rapid solvent transport is matched with very efficient solute mass transfer as fibers are virtually non-porous with respect to the size of the target protein molecules. Lack of porosity of course limits the equilibrium binding capacity of stationary phases. Breakthrough curves and frontal analysis are used to better understand trade-offs between the kinetic and thermodynamic properties as C-CP fibers are applied in preparative situations. Fiber columns packed to different interstitial fraction values affect both the total fiber surface area (e.g., equilibrium binding capacity [EBC]) and the permittivity to flow and mass transport characteristics (e.g., dynamic binding capacity [DBC]). The EBC of the nylon 6 C-CP fibers was found to be 1.30 mg g(-1), with isotherms that were best matched by a Moreau model, showing linearity up to solute concentrations of ∼0.4 mg mL(-1). Isotherms generated under flow conditions were equally well approximated using Langmuir, Freundlich, and Moreau isotherm models. Fairly linear responses were seen up to the maximum load concentration of 1.2 mg mL(-1). Counterintuitively, dynamic studies revealed that conditions of high column porosity yielded a DBC that is ∼70% higher than the EBC. These findings point to potential advantages in terms downstream processing applications, where protein throughput and yield are critical metrics.

Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins

“…Nor would one expect a kinetic advantage in solute/surface transport and binding kinetics. Referring again to studies of BSA on nylon 6 [35], where the primary reactions are with amine end groups, what is seen here is dramatically enhanced capacity due to the presence of tertiary amines in the PEI case. It is not surprising that the extent of the Q m value increases for PEImodified PET columns do not follow the ∼2× increase seen in the results of the ninhydrin tests as a function of PEI reaction time.…”

“…To the best of our knowledge, this is the first study of using PEI modification of PET to affect a stationary phase medium for AEX protein separations. The results presented here suggest that there is a great deal of practical advantage to combining the enhanced selectivity and capacity of the PEI phase and the previously demonstrated throughput and yield characteristics of C-CP fiber columns [7,35].…”

“…5), and creation of isotherms. This approach is preferred over frontal analysis (as has been done previously on C-CP fiber columns [35]) for two reasons. First, load/elution allows for assessment of the entire separation process, inclusive of the primary adsorption step as well as recovery.…”

“…5, and so its value represents the same injection conditions but with the RP elution solvent. The 0.41 mg mL −1 binding capacity value here is not unreasonable versus the DBC for BSA on nylon 6 C-CP fibers of 0.31 mg mL −1 obtained via frontal analysis of breakthrough curves [35]. This value is considered to be the DBC baseline based on the binding kinetics (flow rate) and formation of a monolayer of protein on the available fiber surface.…”

“…The amount of protein retained on the columns was calculated based on the integrated absorbance of the elution peak and comparisons to calibration curve responses. Previous studies have shown that under dynamic (flow) conditions, the binding capacity of C-CP fiber columns is best modeled according to a Langmuir adsorption isotherm [35]. Fig.…”