Suresh Vunnum

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.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two‐column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch‐binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved. Biotechnol. Bioeng. 2008;101: 553–566.

Antibiotics as low-molecular-mass displacers in ion-exchange displacement chromatography

Abstract While the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementtion of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state-of-the-art of displacement chromatography has been the discovery that low-molecular-mass dendritic polymers and protected amino acids can be successfully employed as displacers for protein purification in ion-exchange systems. In this paper, the efficacy of aminoglycosidic antibiotic displacers are investigated for protein purification in cation-exchange systems. The displacers neomycin B and streptomycin A are employed with model feed mixtures containing moderately to very strongly retained proteins. These experiments demonstrate that this new class of low-molecular-mass antibiotic displacers can indeed act as efficient protein displacers. In fact, the displacer neomycin B is the first low-molecular-mass displacer reported i the literature which can readily displace very strongly retained proteins such as lysozyme. In addition to the fundamental interest generaed by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.

Immobilized metal affinity chromatography: Modeling of nonlinear multicomponent equilibrium

A rigorous multicomponent isotherm for preparative immobilized metal affinity chromatography (IMAC) must consider the multipointed nature of adsorption, the possibility of steric hindrance of the stationary phase upon binding of macromolecules, and the role of the mobile-phase modifier. In this paper, the metal affinity interaction chromatography (MAIC) model, a formalism which addresses all three of these issues, is presented. The linear and nonlinear adsorption behavior of proteins as a function of mobile-phase imidazole content is considered. Numerical simulations of nonlinear chromatography, employing MAIC equilibrium, are seen to accurately predict the experimental results in various modes of nonlinear IMAC chromatography. In frontal chromatography, the model is seen to accurately describe the adsorption phenomena, including induced imidazole gradients. In step gradient and displacement chromatography, the results presented in this manuscript demonstrate the ability of the MAIC model to predict multicomponent equilibrium in IMAC systems and establish this model as a powerful tool for studying the operation of IMAC separations.

Immobilized Metal Affinity Chromatography: Displacer Characteristics of Traditional Mobile Phase Modifiers

Imidazole was examined as a potential displacer for protein purification in metal affinity chromatographic systems. A dynamic affinity plot was developed for predicting the elution order and efficacy of displacers in metal affinity displacement chromatography. Theoretical predictions and experimental results indicate that small molecular weight compounds, such as imidazole, with a single coordination site can indeed displace proteins with multiple coordination sites to the surface. In addition, the theory predicts the concentration dependence ofimidazole displacement behavior observed in the experiments. In the presence of additional mobile phase modifiers, imidazole was shown to induce relatively high modifier gradients due to its high affinity and negligible steric factor. N‐protected histidines and tryptophan were also shown to act as protein displacers in IMAC systems. While the N‐protected histidines resulted in relatively sharp displacement boundaries, tryptophan was a less effective displacer, producing significant protein tailing during the separation. The results in this manuscript are expected to have major implications for downstream processing of proteins using IMAC in the displacement mode of operation.

IMAC : Nonlinear elution chromatography of proteins

Nonlinear elution chromatography of proteins employing the traditional mobile phase modulators in IMAC has been studied using the metal affinity interaction model (MAIC) in conjunction with the chromatographic mass transport equations. Results indicate that when the feed is loaded under conditions that foster constant pattern formation, isocratic elution leads to Langmuirian profiles at low mobile phase modulator (MPM) concentrations and a double-plateau profile at high concentrations. A non-self-sharpening protein front during loading, on the other hand, can lead to three plateau profiles or peak splitting and ghost peak formation. Experimental and simulation results on protein separations employing high-affinity modulators suggest that elution with loading in the absence of modulators leads to considerably higher throughputs and protein concentrations due to the displacement effects of the modulator as it migrates through the protein zones. Also, for certain separation problems, the operating conditions can be chosen such that the complex modulator system peaks manifest themselves as spacer displacer; affecting a sharper rear of the earlier eluting protein and an efficient separation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:373-390, 1997.

Nonlinear Multicomponent Gradient Chromatography in Metal Affinity Systems

ABSTRACT In this paper the metal affinity interaction chromatography (MAIC) model is employed in concert with appropriate mass transport equations to study preparative linear gradient chromatography in immobilized metal affinity chromatography (IMAC) systems. The MAIC model accounts for the nonlinear adsorption of proteins and mobile phase modulators (e.g., imidazole), and is shown to accurately predict gradient separations of proteins under overloaded conditions. Experimental and simulation results indicate that the concentration-dependent sorption of imidazole and protein-imidazole interference effects can severely deform linear gradients in IMAC systems. The steric accessibility and displacer characteristics of imidazole together with multicomponent interference effects can lead to unusual protein elution profiles and the spiking of imidazole between the feed components. Due to their ability to act as displacers, these imidazole spikes can sharpen protein tails, decreasing the interface shock layer thi...

Immobilized metal affinity chromatography: Self-sharpening of protein–modulator interfaces in frontal chromatography

Abstract The binding behavior of mobile phase modulators in IMAC is quite different from that of proteins due to their single site interaction and high saturation capacities. Furthermore, stationary phase sites that are sterically shielded for proteins upon macromolecular adsorption are often accessible to mobile phase modifiers. In this paper, the implications of these differences on the self-sharpening of protein–modulator interfaces in frontal chromatography and on the order of elution in binary frontal chromatography are examined. A metal affinity interaction chromatography (MAIC) model is employed to study the behavior of these non-linear systems. The results indicate that for a given protein concentration, there exist a lower and an upper limit of modulator concentration that leads to a self-sharpening protein–modulator interface. Below the lower limit, a single step change in protein concentration can lead to the formation of two protein– modulator interfaces. In addition, protein–modulator binary frontal chromatography in IMAC systems is seen to exhibit dual selectivity reversals. The work presented in this paper has important implications for the determination of protein isotherms in IMAC systems.