Steven M. Cramer

Optimization of preparative ion-exchange chromatography of proteins: linear gradient separations

In this study, the Steric Mass Action (SMA) model of ion exchange is employed in concert with appropriate mass balance equations to predict the separation performance of preparative ion-exchange chromatography. The model is able to accurately predict linear gradient separations of the proteins α-chymotrypsinogen A, cytochrome c, and lysozyme under overloaded conditions. The optimization behavior of preparative ion-exchange chromatography is examined under conditions of baseline resolution and induced sample displacement. This work demonstrates that a simple iterative procedure can be employed to establish optimal gradient conditions for preparative ion-exchange chromatography of proteins. The results also indicate that under appropriate conditions, sample displacement can be employed to dramatically improve the production rate with minor losses in product yield or purity. Linear gradient separations with sample displacement are also less sensitive to the adsorption properties of the feed stream than baseline resolved separations, resulting in simplified methods development.

Membrane chromatographic systems for high-throughput protein separations

Abstract This paper explores the utility of a membrane chromatographic system (MemSep) for analytical and preparative separations of biomolecules. These column systems consist of stacked disks of macroporous cross-linked regenerated cellulose membranes functionalized with ion-exchange moieties. Fluid flow through the macropores of these membranes resultes in rapid mass transport to and from the adsorbent surface. Elution and frontal experiments demonstrated that these systems were relatively insensitive to flow-rate. Linear gradient experiments under analytical conditions indicated that rapid separations could be readily carried out. Preparative-scale separations of proteins on ion-exchange MemSep systems are scaled-up with respect to flow-rate and mass loading with minimal adverse effect on bioproduct purity. A cation-exchange CM MemSep 1010 device was able to concentrate and purify 30 mg and 15 mg of proteins in 3 min when operated in the step and linear gradient modes, respectively. The design of these membrane chromatogrpahic systems enables efficient gradient elution of proteins under elevated flow-rate and mass loading conditions.

Characterization of non-linear adsorption properties of dextran-based polyelectrolyte displacers in ion-exchange systems

Abstract Experimental studies were carried out on the non-linear adsorption properties of dextran-based polyelectrolytes in anion- and cation-exchange chromatographic systems. By monitoring both the induced salt gradients and sequential breakthrough fronts, parameters were determined for use in a Steric Mass Action (SMA) model of non-linear ion-exchange chromatography. These parameters include: total ion capacity of the columns, characteristic charge, steric factor, equilibrium constant, and maximum adsorptive capacity for each of the polyelectrolytes. In addition the number of functional groups were determined by elemental analysis. The values of the SMA parameters were found to be independent of salt and polyelectrolyte bulk phase compositions. Parameters were also determined for a variety of proteins. Experimental isotherms for the polyelectrolytes and proteins were compared with those simulated by the SMA model. Finally, the implications of polyelectrolyte adsorption properties with respect to their ability to act as efficient displacers in ion-exchange displacement systems are discussed.

Ion-exchange displacement chromatography of proteins: Dextran-based polyelectrolytes as high affinity displacerss

Abstract Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.

Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

While the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of biopharmaceuticals, a major obstacle to the implementation of displacement chromatography has been the lack of appropriate displacer compounds. All protein displacement separations reported to date have employed relatively high-molecular-mass (> 2000) polyelectrolyte displacers. In this paper, results are presented on the discovery that low-molecular-mass dendritic polymers can be successfully employed as efficient displacers for protein purification in ion-exchange systems. Pentaerythritol-based dendritic polyelectrolytes ranging in molecular mass from 480 to 5100 were investigated as potential displacers for the purification of proteins in cation-exchange systems. The adsorption properties of these dendrimers were investigated using the steric mass action (SMA) model of non-linear ion-exchange chromatography. An analysis of the resulting SMA parameters using a dynamic affinity plot indicated that these dendrimers should have sufficient affinity to act as protein displacers. Displacement separations of protein mixtures in cation-exchange systems were carried out using zero-, first- and second-generation dendrimers. These experiments demonstrate that this new class of dendritic polyelectrolytes can indeed act as efficient protein displacers. The ability of a low-molecular-mass compound such as the “zero”-generation dendrimer (Mr 480) to displace proteins is very significant in that it represents a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion-exchange systems. In addition to the fundamental interest generated by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.

Theoretical optimization of operating parameters in non-ideal displacement chromatography

Abstract A mathematical model was developed for the simulation of non-ideal displacement chromatography. The model incorporates finite mass transport to the solid adsorbent by using a linear driving force approximation with a coupled external film and internal pore mass transfer coefficient. Equilibrium adsorption at the fluid—solid interface is described using competitive langmuirian adsorption isotherms. A finite difference numerical technique was employed to approximate the system of coupled, non-linear partial differential equations. The model was used to simulate the effluent concentration profiles under various displacement chromatographic conditions. The effects of axial dispersion and finite mass transport were examined by varying the Peclet and Stanton numbers, respectively. Slow mass transfer rates were shown to have a dispersive effect on the shock waves generated in displacement chromatography, resulting in greater zone overlap. Constant pattern formation was observed under non-ideal conditions. The throughput obtained in displacement chromatography was examined as a function of feed load, flow velocity, and displacer concentration. For non-ideal systems, the throughput was shown to exhibit a maximum at unique values of these operating parameters. The effects of particle diameter and solute diffusivity on the throughput were also examined. Model predictions indicate that the use of large particles could be detrimental to the performance of displacement systems when high velocities are employed. For macromolecular separations by displacement chromatography, small particles are required regardless of the linear velocity. The model presented here is a useful tool for the optimization and scale-up of displacement chromatographic processes.

Displacement chromatography of biomolecules

Displacement chromatography was used for the preparative-scale separation of peptides, antibiotics, and proteins. The feed components were both purified and concentrated during the separation processes. The components of a peptide mixture were separated on a reverse-phase analytical column using 2-(2-butoxyethoxy) ethanol as the displacer. The use of organic modifiers in the carrier along with an elevated column temperature of 45 degrees C enabled the efficient separation of relatively hydrophobic peptides by displacement chromatography. In addition, the throughput of the process was significantly increased by carrying out the separation at an elevated flow-rate with no adverse effect on product purity. The antibiotic cephalosporin C was isolated from impurities in a fermentation broth using 2-(2-butoxyethoxy)ethanol as the displacer along with a step change in column temperature. The proteins cytochrome c and lysozyme were purified on a weak cation-exchanger column using cationic polymers as the displacers. While polymers of 60 and 20 kilodaltons were both found to be good displacers for these proteins, only the lower molecular weight polymer was readily removed from the column by standard regeneration techniques.

Modeling non-linear elution of proteins in ion-exchange chromatography

Abstract The problem of non-linear elution of a band of protein in isocratic ion-exchange chromatography leads to a pair of coupled non-linear partial differential equations. The equilibrium may be modeled using the Steric Mass Action (SMA) model of ion exchange, which treats both the salt dependence of protein adsorption and the steric shielding present under non-linear conditions. Neglecting axial dispersion, a model of ideal chromatography is formulated that may be solved by the method of characteristics. The predictions of this relatively simple model are shown to agree with experimental results concerning the non-linear elution of cytochrome c in a strong cation-exchange column. Of particular interest is the existence of two plateaus in the solution of this problem for large injection volumes. While this result cannot be understood or predicted on the basis of the traditional Langmuir isotherm or other currently available descriptions of adsorption, the chromatographic model presented in this work makes this otherwise anomalous result clear. Further, the use of such a model during parameter estimation is discussed.

Parallel synthesis and screening of polymers for nonviral gene delivery

We describe the parallel synthesis and in vitro evaluation of a cationic polymer library for the discovery of nonviral gene delivery vectors. The library was synthesized based on the ring-opening polymerization reaction between epoxide groups of diglycidyl ethers and the amines of (poly)amines. Parallel screening of soluble library constituents led to the identification of lead polymers with high DNA-binding efficacies. Transfection efficacies of lead polymers were evaluated using PC3-PSMA human prostate cancer cells and murine osteoblasts in the absence and presence of serum. In vitro experiments resulted in the identification of a candidate polymer that demonstrated significantly higher transfection efficacies and lower cytotoxicities than poly(ethyleneimine) (pEI), the current standard for polymeric transfection agents. In addition, polymers that demonstrated moderately higher and comparable transfection efficacies with respect to pEI were also identified. Our results demonstrate that high-throughput synthesis and screening of polymers is a powerful approach for the identification of novel nonviral gene delivery agents.

Characterization and modeling of monolithic stationary phases: application to preparative chromatography

A methodology for characterizing and modeling preparative separations on monolithic ion-exchange stationary phases is presented. A dimensionless group analysis was carried out to determine the relative importance of mass transfer and kinetic resistances on this stationary phase. In contrast to conventional beaded morphologies, the continuous bed stationary phase was found to possess enhanced mass transport properties resulting in kinetic resistance as the dominant non-ideality. Accordingly, a reaction-dispersive steric-mass action formalism was successfully utilized for simulating preparative displacement chromatography on this resin. Since kinetics were found to be important on this column morphology, mobile phase salt concentration was found to be an important variable during displacement chromatography on this stationary phase. An increase in the mobile phase salt concentration was found to significantly improve the displacement separation of a model protein mixture. The formalism presented in this paper provides a better understanding of preparative chromatography in monolithic resin systems and a means of simulating separations on this class of chromatographic stationary phases.