Why nanofibers are a good adsorptive surface – fundamental understanding and industrial applications for mAb bioprocessing

Over the years, chromatography has proven to be a powerful and versatile technique for the purification of high value biotherapeutics. Yet, today’s preparative chromatography of biologics still, in principle, looks the same as it did several decades ago. Any improvements made have been incremental; constrained by the stationary phase format (porous beads), associated column size (bed height and pressure drop), and historical modes of operation. To address future manufacturing challenges such as high cost of goods, diversity in product portfolios, market dynamics and manufacturing flexibility, new, more radical approaches to the development of chromatography materials and towards associated modes of operations are needed. With the biotechnology industry maturing, wide spread adoption of new high tech tools/products such as high throughput analytics, automated process control, single use materials and real time data analysis is already taking place, which in turn will lead towards revisiting and a subsequent improvement of how chromatography will be operated in the future. Examples of such improvements that are already considered include high productivity operations such as simulated moving bed and rapid, or extreme, cycling regimes. Please click Additional Files below to see the full abstract

Advanced Analysis of Biosensor Data for SARS-CoV-2 RBD and ACE2 Interactions

The traditional approach for analyzing interaction data from biosensors instruments is based on the simplified assumption that also larger biomolecules interactions are homogeneous. It was recently reported that the human receptor angiotensin-converting enzyme 2 (ACE2) plays a key role for capturing SARS-CoV-2 into the human target body, and binding studies were performed using biosensors techniques based on surface plasmon resonance and bio-layer interferometry. The published affinity constants for the interactions, derived using the traditional approach, described a single interaction between ACE2 and the SARS-CoV-2 receptor binding domain (RBD). We reanalyzed these data sets using our advanced four-step approach based on an adaptive interaction distribution algorithm (AIDA) that accounts for the great complexity of larger biomolecules and gives a two-dimensional distribution of association and dissociation rate constants. Our results showed that in both cases the standard assumption about a single interaction was erroneous, and in one of the cases, the value of the affinity constant K-D differed more than 300% between the reported value and our calculation. This information can prove very useful in providing mechanistic information and insights about the mechanism of interactions between ACE2 and SARS-CoV-2 RBD or similar systems

Modeling and Analysis of the Dynamic Behavior of Mechanisms That Result in the Development of Inner Radial Humps in the Concentration of a Single Adsorbate in the Adsorbed Phase of Porous Adsorbent Particles Observed in Confocal Scanning Laser Microscopy Experiments: Diffusional Mass Transfer and Adsorption in the Presence of an Electrical Double Layer

A theoretical model for adsorption of a single charged adsorbate that accounts for the presence of an electrical double layer in the pores of adsorbent particles is constructed and solved. The dynamic behavior of the mechanisms of the model can result in the development of inner radial humps (concentration rings) in the concentration of a single charged analyte (adsorbate) in the adsorbed phase of porous adsorbent particles. The results of the present work demonstrate the implication of the concept regarding the effect of the presence of an electrical double layer in the pores of adsorbent particles and the induced interactions between the electrostatic potential distribution and the mechanisms of mass transport of the species by diffusion, electrophoretic migration, and adsorption. Furthermore, the mechanisms of the model could explain qualitatively the development of the concentration ring (hump) observed in confocal scanning laser microscopy experiments

Molecular Dynamics Simulation Studies of the Transport and Adsorption of a Charged Macromolecule Onto a Charged Adsorbent Solid Surface Immersed in an Electrolytic Solution

Molecular dynamics simulations were performed in order to study the transport and adsorption of a charged macromolecule (desmopressin) onto a charged solid surface in an electrolytic solution. The strong Coulombic interaction from the charged solid surface represents the major force for accelerating, orienting, entrapping in the electrical double layer, and adsorbing the macromolecule onto the charged solid surface. The macromolecule is flattened as it approaches the charged surface, giving rise to a stronger surface exclusion effect that shields surface sites. when adsorbed, the macromolecule is restrained by a surface interaction more than one hundred times stronger than the thermal energy, of which 99.8% results from the strong dominant Coulombic interaction, and trapped by a hydration layer adjacent to the surface. This leads to zero lateral displacement of the adsorbed macromolecule and indicates that surface diffusion is a physically implausible mechanism in similar systems. Explicit solvent is required for realistic representation of the macromolecular structure and the surface interaction energy. The adsorbed macromolecule also decreased the electrostatic potential gradient perpendicular to the charged solid surface and introduced additional electrostatic potential gradients laterally. The results obtained from the molecular dynamics simulations confirm the importance of electrophoretic migration and support the physical mechanisms used in a macroscopic continuum model that predicts an overshoot in the concentration of a charged macromolecule in the adsorbed phase under certain conditions of pH and ionic strength

Molecular Dynamics Simulation Studies of the Conformation and Lateral Mobility of a Charged Adsorbate Biomolecule: Implications for Estimating the Critical Value of the Radius of a Pore in Porous Media

The conformations, the values of the lateral transport coefficient of a charged biomolecule (desmopressin) in the adsorbed layer and in the liquid layers above the adsorbed layer, the potential energies of the interaction between the biomolecules located in different liquid layers with the charged solid surface and with the biomolecules in the adsorbed layer, the potential energies of the interaction between water molecules in the hydration layers surrounding the conformations of the biomolecules in different layers, as well as the structure and number of hydration layers between the different conformations of desmopressin, were determined by molecular dynamics simulation studies. The results show that the lateral mobility of the adsorbed desmopressin is approximately equal to zero and the value of the lateral transport coefficient of the biomolecule in the liquid layers located above the adsorbed layer increases as the distance of the liquid layer from the charged solid surface increases. But the values of the lateral transport coefficient of the biomolecule in the liquid layers above the adsorbed layer are lower in magnitude than the value of the transport coefficient of desmopressin along the direction normal to the charged solid surface in the liquid phase located above the vacant charged sites of the solid surface, and these differences in the values of the transport coefficients have important implications with respect to the replenishment of the biomolecules in the inner parts of a channel (pore), the overall rate of adsorption, and the form of the constitutive equations that would have to be used in macroscopic models to describe the mechanisms of mass transfer and adsorption in the pores of adsorbent media. Furthermore, a novel method is presented in this work that utilizes the information about the sizes of the conformations of the biomolecule in the adsorbed layer and in the liquid layers above the adsorbed layer along the direction that is normal to the charged solid surface, as well as the number and size of the hydration layers along the same direction, and could be used to estimate the value of the lower bound of the linear characteristic dimension of a pore (i.e., pore radius) in porous adsorbent media (e.g., porous adsorbent particles; skeletons of porous monoliths) in order to realize effective transport and overall adsorption rate

Construction by Molecular Dynamics Modeling and Simulations of the Porous Structures Formed by Dextran Polymer Chains Attached on the Surface of the Pores of a Base Matrix: Characterization of Porous Structures

Significant increases in the separation of bioactive molecules by using ion-exchange chromatography are realized by utilizing porous adsorbent particles in which the affinity group/ligand is linked to the base matrix of the porous particle via a polymeric extender. To study and understand the behavior of such systems, the M3B model is modified and used in molecular dynamics (MD) simulation studies to construct porous dextran layers on the surface of a base matrix, where the dextran polymer chains and the surface are covered by water. Two different porous polymer layers having 25 and 40 monomers per main polymer chain of dextran, respectively, are constructed, and their three-dimensional (3D) porous structures are characterized with respect to porosity, pore size distribution, and number of conducting pathways along the direction of net transport. It is found that the more desirable practical implications with respect to structural properties exhibited by the porous polymer layer having 40 monomers per main polymer chain, are mainly due to the higher flexibility of the polymer chains of this system, especially in the upper region of the porous structure. The characterization and analysis of the porous structures have suggested a useful definition for the physical meaning and implications of the pore connectivity of a real porous medium that is significantly different than the artificial physical meaning associated with the pore connectivity parameter employed in pore network models and whose physical limitations are discussed; furthermore, the methodology developed for the characterization of the three-dimensional structures of real porous media could be used to analyze the experimental data obtained from high-resolution noninvasive three-dimensional methods like high-resolution optical microscopy. The MD modeling and simulations methodology presented here could be used, considering that the type and size of affinity group/ligand as well as the size of the biomolecule to be adsorbed onto the affinity group/ligand are known, to construct different porous dextran layers by varying the length of the polymeric chain of dextran, the number of attachment points to the base matrix, the degree of side branching, and the number of main polymeric chains immobilized per unit surface area of base matrix. After the characterization of the porous structures of the different porous dextran layers is performed, then only a few promising structures would be selected for studying the immobilization of adsorption sites on the pore surfaces and the subsequent adsorption of the bioactive molecules onto the immobilized affinity groups/ligands

Analysis and Parametric Sensitivity of the Behavior of Overshoots in the Concentration of a Charged Adsorbate in the Adsorbed Phase of Charged Adsorbent Particles: Practical Implications for Separations of Charged Solutes

In this work, an analysis of the parametric sensitivity of the overshoot in the concentration of the adsorbate in the adsorbed phase, which occurs under certain conditions during an ion-exchange adsorption process, is presented and used to suggest practical implications of the concentration overshoot phenomenon on operational policies and configurations of chromatographic columns and finite bath adsorption systems. The results presented in this work demonstrate and explain how the development of an overshoot in the concentration of the adsorbate in the adsorbed phase could be enhanced or suppressed by (i) varying the diffusion coefficient, D3, of the adsorbate relative to the diffusion coefficients, D1 and D2, of the cations and anions, respectively, of the background/buffer electrolyte, (ii) altering the initial surface charge density, δ0, of the charged adsorbent particles, (iii) varying the Debye length, λ, and (iv) changing the initial concentration, Cd30, of the adsorbate in the bulk liquid of the finite bath. The influence of the pH and ionic strength, I∞, of the liquid solution on the development of an overshoot in the concentration of the adsorbate in the adsorbed phase is also presented and discussed through the relationships of these parameters to δ0 and λ, respectively. Furthermore, a detailed explanation of the effects of each parameter on the interplay between the diffusive and electrophoretic molar fluxes, as well as on the structure and functioning of the electrical double layer, which are responsible for the concentration overshoot phenomenon, is presented