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.

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization.

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by d-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields.

Virus clearance validation across continuous capture chromatography.

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.

Model-assisted process characterization and validation for a continuous two-column protein A capture process.

In this study we introduce three process characterization approaches toward validation of continuous twin-column capture chromatography (CaptureSMB), referred to as "standard," "model assisted," and "hybrid." They are all based on a traditional risk-based approach, using process description, risk analysis, design-of-experiments (DoE), and statistical analysis as essential elements. The first approach, the "standard" approach uses a traditional experimental DoE to explore the design space of the high-ranked process parameters for the continuous process. Due to the larger number of process parameters in the continuous process, the DoE is extensive and includes a larger number of experiments than an equivalent DoE of a single column batch capture process. In the investigated case, many of the operating conditions were practically infeasible, indicating that the design space boundaries had been chosen inappropriately. To reduce experimental burden and at the same time enhance process understanding, an alternative "model assisted" approach was developed in parallel, employing a chromatographic process model to substitute experimental runs by computer simulations. Using the "model assisted" approach only experimental conditions that were feasible in terms of process yield constraints (>90%) were considered for statistical analysis. The "model assisted" approach included an optimization part that identified potential boundaries of the design space automatically. In summary, the "model assisted" approach contributed to increased process understanding compared to the "standard" approach. In this study, a "hybrid" approach was also used containing the general concepts of the "standard" approach but substituting a number of its experiments by computer simulations. The presented approaches contain essential elements of the Food and Drug Administration's process validation guideline.

Role of the gradient slope during the product internal recycling for the multicolumn countercurrent solvent gradient purification of PEGylated proteins.

Protein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an efficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764-10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three different gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing.

Design and economic investigation of a Multicolumn Countercurrent Solvent Gradient Purification unit for the separation of an industrially relevant PEGylated protein.

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility.

Coronavirus (COVID-19) and Racial Disparities: a Perspective Analysis.

Health disparity refers to systematic differences in health outcomes between groups and communities based on socioeconomic isolation. In the USA, health disparities among minority groups, especially African Americans, limit their access to quality medical care and other beneficial resources and services. Presently, the novel coronavirus (COVID-19) highlights the extreme healthcare challenges that exist in the African American and other minority communities in the USA. African Americans are dying at a rate nearly four times higher than the national average. With inadequate access to quality healthcare, viable resources, and information, COVID-19 will continue to have a disastrous effect on African American communities. This communication provides a brief overview of the health inequalities resulting in African Americans dying disproportionately during the COVID-19 pandemic.

Model based strategies towards protein A resin lifetime optimization and supervision.

The high cost of protein A resins drives the biopharmaceutical industry to maximize its lifetime, which is limited by several processes, usually referred to as resin aging. In this work, two model based strategies are presented, aiming to control and improve the resin lifetime. The first approach, purely statistical, enables qualitative monitoring of the column state and prediction of column performance indicators (e.g. yield, purity and dynamic binding capacity) from chromatographic on-line data (e.g. UV signal). The second one, referred to as hybrid modeling, is based on a lumped kinetic model, which includes two aging parameters fitted on several resin cycling experimental campaigns with varying cleaning procedures (CP). The first aging parameter accounts for binding capacity deterioration (caused by ligand degradation, leaching, and pore occlusion), while the second accounts for a decreased mass transfer rate (mainly caused by fouling). The hybrid model provides important insights into the prevailing aging mechanism as a function of the different CPs. In addition, it can be applied to model based CP optimization and yield forecasting with the capability of state estimation corrections based on on-line process information. Both approaches show promising results, which could help to significantly extend the resin lifetime. This comes along with increased understanding, reduced experimental effort, decreased cost of goods, and improved process robustness.

Biomanufacturing evolution from conventional to intensified processes for productivity improvement: a case study.

Process intensification has shown great potential to increase productivity and reduce costs in biomanufacturing. This case study describes the evolution of a manufacturing process from a conventional processing scheme at 1000-L scale (Process A, n = 5) to intensified processing schemes at both 1000-L (Process B, n = 8) and 2000-L scales (Process C, n = 3) for the production of a monoclonal antibody by a Chinese hamster ovary cell line. For the upstream part of the process, we implemented an intensified seed culture scheme to enhance cell densities at the seed culture step (N-1) prior to the production bioreactor (N) by using either enriched N-1 seed culture medium for Process B or by operating the N-1 step in perfusion mode for Process C. The increased final cell densities at the N-1 step allowed for much higher inoculation densities in the production bioreactor operated in fed-batch mode and substantially increased titers by 4-fold from Process A to B and 8-fold from Process A to C, while maintaining comparable final product quality. Multiple changes were made to intensify the downstream process to accommodate the increased titers. New high-capacity resins were implemented for the Protein A and anion exchange chromatography (AEX) steps, and the cation exchange chromatography (CEX) step was changed from bind-elute to flow-through mode for the streamlined Process B. Multi-column chromatography was developed for Protein A capture, and an integrated AEX-CEX pool-less polishing steps allowed semi-continuous Process C with increased productivity as well as reductions in resin requirements, buffer consumption, and processing times. A cost-of-goods analysis on consumables showed 6.7-10.1 fold cost reduction from the conventional Process A to the intensified Process C. The hybrid-intensified process described here is easy to implement in manufacturing and lays a good foundation to develop a fully continuous manufacturing with even higher productivity in the future.

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.

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.

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.

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.