Continued insights into virus clearance validation across continuous capture chromatography.

Multi-column capture chromatography (MCC) has gained increased attention lately due to the significant economic and process-related advantages it offers compared to traditional batch mode chromatography. However, for wide adoption of this technology in the clinical and commercial space, it requires scalable models for viral validation. In this study, additional viral validation studies were conducted under cGLP guidelines to assess retro-(X-MuLV) and parvo-virus (minute virus of mice) clearance across twin-column continuous capture chromatography (CaptureSMB) to supplement work previously performed. A surrogate model was validated using standard batch mode chromatography equipment based on flow path modifications to mimic the loading strategy employed in CaptureSMB. In addition, aged resin was used in this surrogate format to assess the impact of resin lifetime on viral clearance during continuous capture operation. The impact of column loading was also explored to shed light on the viral clearance mechanisms of protein A chromatography in overloading conditions. The proposed approach greatly simplifies MCC virus validation studies, and provides a robust strategy for regulatory filing of continuous biomanufacturing processes.

Model assisted comparison of Protein A resins and multi-column chromatography for capture processes.

Effect of particle size (85µm vs. 50µm) on the performance of continuous capture chromatography using Protein A affinity was evaluated in combination with varied feed titers, loading flow rates and target breakthrough using a Design of Experiments (DoE) approach. In comparison to previous studies, higher cell culture titers on the order of 5-15 g/L, relevant to current high productivity industrial cell lines, were evaluated. Further, three modes of capture continuous chromatography were included in the DoE: single column batch, 2-column CaptureSMB and 4-column periodic counter-current chromatography (PCC). The breakthrough percentage at the outlet of the first column being loaded showed the most significant impact on process performance, confirming the advantage of multi-column over batch chromatography processes. Out of the two resins, the one with smaller particle size displayed significantly better performance. To verify and generalize these results, a shrinking core model for protein A chromatography has been developed and validated. The model was used to optimize the processes with respect to capacity utilization (load per cycle) and productivity (load per time). The smaller particle size resin (50µm) produced steeper breakthrough curves and allowed for better capacity utilization at any given productivity value. The improvement in loading was around 15% on average in comparison to the 85µm bead size in spite of the ligand density being same. The 50µm resin also allowed for higher maximum productivity values compared to the 85µm resin (improvements of 25-50%, depending on the process), despite lower maximum flow rate due to increased pressure drop. In addition, it is worth noting that recovery and regeneration rather than the maximum flow rate (pressure drop) became the limiting factor for process optimization in almost all considered scenarios.

Ionic strength-dependent changes in tentacular ion exchangers with variable ligand density. II. Functional properties.

The effect of ligand density was studied on protein adsorption and transport behavior in tentacular cation-exchange sorbents at different ionic strengths. Results were obtained for lysozyme, lactoferrin and a monoclonal antibody (mAb) in order to examine the effects of protein size and charge. The combination of ligand density and ionic strength results in extensive variability of the static and dynamic binding capacities, transport rate and binding affinity of the proteins. Uptake and elution experiments were performed to quantify the transport behavior of selected proteins, specifically to estimate intraparticle protein diffusivities. The observed trend of decreasing uptake diffusivities with an increase in ligand density was correlated to structural properties of the ligand-density variants, particularly the accessible porosity. Increasing the ionic strength of the equilibration buffer led to enhanced mass transfer during uptake, independent of the transport model used, and specifically for larger proteins like lactoferrin and mAb, the most significant effects were evident in the sorbent of the highest ligand density. For lysozyme, higher ligand density leads to higher static and dynamic binding capacities whereas for lactoferrin and the mAb, the binding capacity is a complex function of accessible porosity due to ionic strength-dependent changes. Ligand density has a less pronounced effect on the elution rate, presumably due to ionic strength-dependent changes in the pore architecture of the sorbents.

Characterization of dextran-grafted hydrophobic charge-induction resins: Structural properties, protein adsorption and transport.

The structural and functional properties of a series of dextran-grafted and non-grafted hydrophobic charge-induction chromatographic (HCIC) agarose resins were characterized by macroscopic and microscopic techniques. The effects of dextran grafting and mobile phase conditions on the pore dimensions of the resins were investigated with inverse size exclusion chromatography (ISEC). A significantly lower pore radius (17.6nm) was found for dextran-grafted than non-grafted resins (29.5nm), but increased salt concentration would narrow the gap between the respective pore radii. Two proteins, human immunoglobulin G (hIgG) and bovine serum albumin (BSA), were used to examine the effect of protein characteristics. The results of adsorption isotherms showed that the dextran-grafted resin with high ligand density had substantially higher adsorption capacity and enhanced the salt-tolerance property for hIgG, but displayed a significantly smaller benefit for BSA adsorption. Confocal laser scanning microscopy (CLSM) showed that hIgG presented more diffuse and slower moving adsorption front compared to BSA during uptake into the resins because of the selective binding of multiple species from polyclonal IgG; polymer-grafting with high ligand density could enhance the rate of hIgG transport in the dextran-grafted resins without salt addition, but not for the case with high salt and BSA. The results indicate that microscopic analysis using ISEC and CLSM is useful to improve the mechanistic understanding of resin structure and of critical functional parameters involving protein adsorption and transport, which would guide the rational design of new resins and processes.

Determinants of protein elution rates from preparative ion-exchange adsorbents.

The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport.

Adsorption behavior in the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) was characterized using methods to assess, quantitatively and qualitatively, the dynamics of protein uptake as well as static adsorption as a function of ionic strength and protein concentration using several model proteins. The three exchangers studied all presented relatively high adsorptive capacities under low ionic strength conditions, comparable to commercially available resins containing polymer functionalization aimed at increasing that particular characteristic. The strong cation- and anion-exchange moieties showed higher sensitivity to increasing salt concentrations, but protein affinity on the salt-tolerant STAR AX HyperCel exchanger remained strong at ionic strengths normally used in downstream processing to elute material fully during ion-exchange chromatography. Very high uptake rates were observed in both batch kinetics experiments and time-series confocal laser scanning microscopy, suggesting low intraparticle transport resistances relative to external film resistance, even at higher bulk protein concentrations where the opposite is typically observed. Electron microscopy imaging of protein adsorbed phases provided additional insight into particle structure that could not be resolved in previous work on the bare resins.

Characterization of cross-linked cellulosic ion-exchange adsorbents: 1. Structural properties.

The structural characteristics of the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) were assessed using methods to gauge the pore dimensions and the effect of ionic strength on intraparticle architecture. Inverse size exclusion chromatography (ISEC) was applied to the S and STAR AX HyperCel derivatives. The theoretical analysis yielded an average pore radius for each material of about 5nm, with a particularly narrow pore-size distribution. Electron microscopy techniques were used to visualize the particle structure and relate it to macroscopic experimental data. Microscopy of Q and STAR AX HyperCel anion exchangers presented some qualitative differences in pore structure that can be attributed to the derivatization using conventional quaternary ammonium and salt-tolerant ligands, respectively. Finally, the effect of ionic strength was studied through the use of salt breakthrough experiments to determine to what extent Donnan exclusion plays a role in restricting the accessible pore volume for small ions. It was determined that Donnan effects were prevalent at total ionic strengths (TIS) less than 150mM, suggesting the presence of a ligand-containing partitioning volume within the pore space.