Modeling scalability of impurity precipitation in downstream biomanufacturing.

Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale-down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing. Scaling parameters such as agitation and vessel geometry were investigated, with the precipitate amount and particle size distribution (PSD) characterized via turbidity and flow imaging microscopy. Qualitative analysis of the data shows that maintaining a consistent energy dissipation rate (EDR) could be used for approximate scaling of vessel geometry and agitator speeds in the absence of more detailed simulation. For a more rigorous approach, however, agitation was simulated via computational fluid dynamics (CFD) and these results were applied alongside a population balance model to simulate the trajectory of the size distribution of precipitate. CFD results were analyzed within a framework of a two-compartment mixing model comprising regions of high- and low-energy agitation, with material exchange between the two. Rate terms accounting for particle formation, growth and breakage within each region were defined, accounting for dependence on turbulence. This bifurcated model was successful in capturing the variability in particle sizes over time across scales. Such an approach enhances the mechanistic understanding of impurity precipitation and provides additional tools for model-assisted prediction for process scaling.

Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography.

Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes. Column chromatography reveals that the protein A elution tail can have a relatively high concentration of aggregates, which corroborates analogous observations from recent HCP studies. Similar measurements in flow-through AEX chromatography show that relatively large aggregates that harbor HCPs and that persist into the protein A eluate can be retained to an extent that appears to depend primarily on the resin surface chemistry. The total aggregate mass fraction of both protein A eluate pools (∼ 2.4 - 3.6%) and AEX flow-through fractions (∼ 1.5 - 3.2%) correlates generally with HCP concentrations measured using enzyme-linked immunosorbent assay (ELISA) as well as the number of HCPs that may be identified in proteomic analysis. This suggests that quantification of the aggregate mass fraction may serve as a convenient albeit imperfect surrogate for informing early process development decisions regarding HCP clearance strategies.

Engineering protein A ligands to mitigate antibody loss during high-pH washes in protein A chromatography.

Protein A chromatography is a workhorse in monoclonal antibody (mAb) manufacture since it provides effective separation of mAbs from impurities such as host-cell proteins (HCPs) in a single capture step. HCP clearance can be aided by the inclusion of a wash step prior to low-pH elution. Although high-pH washes can be effective in removing additional HCPs from the loaded column, they may also contribute to a reduced mAb yield. In this work we show that this yield loss is reflected in a pH-dependent variation of the equilibrium binding capacity of the protein A resin, which is also observed for the capacity of the Fc fragments alone and therefore not a result of steric interactions involving the Fab fragments in the intact mAbs. We therefore hypothesized that the high-pH wash loss was due to protonation or deprotonation of ionizable residues on the protein A ligand. To evaluate this, we applied a rational protein engineering approach to the Z domain (the Fc-binding component of most commercial protein A ligands) and expressed engineered mutants in E. coli. Biolayer interferometry and affinity chromatography experiments showed that some of the Z domain mutants were able to mitigate wash loss at high pH while maintaining similar binding characteristics at neutral pH. These experiments enabled elucidation of the roles of specific interactions in the Z domain - Fc complex, but more importantly offer a route to ameliorating the disadvantages of high-pH washes in protein A chromatography.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Comparison of host cell protein removal by depth filters with diatomaceous earth and synthetic silica filter aids using model proteins.

A number of studies have demonstrated that depth filtration can provide significant adsorptive removal of host cell proteins (HCP), but there is still considerable uncertainty regarding the underlying factors controlling HCP binding. This study compared the binding characteristics of two fine grade depth filters, the X0SP (polyacrylic fiber with a synthetic silica filter aid) and X0HC (cellulose fibers with diatomaceous earth (DE) as a filter aid), using a series of model proteins with well-defined physical characteristics. Protein binding to the X0SP filter was dominated by electrostatic interactions with greatest capacity for positively-charged proteins. In contrast, the X0HC filter showed greater binding of more hydrophobic proteins although electrostatic interactions also played a role. In addition, ovotransferrin showed unusually high binding capacity to the X0HC, likely due to interactions with metals in the DE. Scanning Electron Microscopy with Energy Dispersive Spectroscopy was used to obtain additional understanding of the binding behavior. These results provide important insights into the physical phenomena governing HCP binding to both fully synthetic and natural (cellulose + DE) depth filters.

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.

Ceramic hydroxyapatite chromatography plays a critical role in bispecific antibody purification process for impurity removal.

Background: Significant challenges exist in downstream purification of bispecific antibodies (BsAbs) due to the complexity of BsAb architecture. A unique panel of mispaired species can result in a higher level of product-related impurities. In addition to process-related impurities such as host cell proteins (HCPs) and residual DNA (resDNA), these product-related impurities must be separated from the targeted BsAb product to achieve high purity. Therefore, development of an efficient and robust chromatography purification process is essential to ensure the safety, quality, purity and efficacy of BsAb products that consequently meet regulatory requirements for clinical trials and commercialization. Methods: We have developed a robust downstream BsAb process consisting of a mixed-mode ceramic hydroxyapatite (CHT) chromatography step, which offers unique separation capabilities tailored to BsAbs, and assessed impurity clearance. Results: We demonstrate that the CHT chromatography column provides additional clearance of low molecular weight (LMW) and high molecular weight (HMW) species that cannot be separated by other chromatography columns such as ion exchange for a particular BsAb, resulting in ≥98% CE-SDS (non-reduced) purity. Moreover, through Polysorbate-80 (PS-80) spiking and LC-MS HCP assessments, we reveal complete clearance of potential PS-80-degrading HCP populations in the CHT eluate product pool. Conclusions: In summary, these results demonstrate that CHT mixed-mode chromatography plays an important role in separation of product- and process-related impurities in the BsAb downstream process.

Use of Monte Carlo simulations for improved facility fit planning in downstream biomanufacturing and technology transfer.

Biologics manufacturing is capital and consumable intensive with need for advanced inventory planning to account for supply chain constraints. Early-stage process design and technology transfer are often challenging due to limited information on process variability regarding bioreactor titer, process yield, and product quality. Monte Carlo (MC) methods offer a stochastic modeling approach for process optimization where probabilities of occurrence for process inputs are incorporated into a deterministic model to simulate more likely scenarios for process outputs. In this study, we explore MC simulation-based design of a monoclonal antibody downstream manufacturing process. We demonstrate that this probabilistic approach offers more representative outcomes over the conventional worst-case approach where the theoretical minimum and maximum values of each process parameter are used without consideration for their probability of occurrence. Our work demonstrates case studies on more practically sizing unit operations to improve consumable utilization, thereby reducing manufacturing costs. We also used MC simulations to minimize process cadence by constraining the number of cycles per unit operation to fit facility preferences. By factoring in process uncertainty, we have implemented MC simulation-based facility fit analyses to efficiently plan for inventory when accounting for process constraints during technology transfer from lab-scale to clinical or commercial manufacturing.

Effect of solution condition on the binding behaviors of monoclonal antibody and fusion protein therapeutics in Protein A chromatography.

Recent development in Protein A chromatography has adopted wash buffers with a wide range of pH to improve the removal of process-related impurities, e.g., host cell proteins (HCPs), from Fc-based target biological products. Thus, it is important to investigate the pH effect on the binding behaviors of target products of various properties on Protein A resins. Here we performed column breakthrough experiments for two monoclonal antibodies (mAbs) and one Fc-fusion protein on two Protein A resins from pH 4 to pH 10, and the experimental data was analyzed using a mechanistic model to obtain isotherm and mass transfer parameters. The two mAbs exhibited relatively high dynamic binding capacity (DBC) at 10% breakthrough of 43 - 67 g/L at pH 6-9 followed by a ∼30% decrease from pH 9 to pH 10; while the Fc-fusion protein reached the highest DBC at 10% breakthrough of 10 - 17 g/L at pH 5 and thereafter the capacity gradually reduced. Model analyses revealed that the two mAbs had higher maximum binding capacity by 2 - 5 fold but weaker binding affinity (12 - 64%) than the Fc-fusion protein from pH 5 to pH 9. For the three molecules, similar patterns of the pH impact were observed on the two Protein A resins with the Jetted A50 resin showing generally higher DBC and stronger binding affinity than the MabSelect SuRe LX resin. Additionally, an inverse relationship between the binding affinity and surface diffusivity was observed for both resins. Besides obtaining the isotherm parameters from the column breakthrough data, a direct batch equilibrium measurement showed comparable trend in these parameters with relatively more scattered values due to the inherent uncertainties to accurately determine the initial slope of the isotherm in highly favorable adsorption conditions. Finally, isothermal titration calorimetry (ITC) results revealed that the measured binding affinity using free Protein A ligand was stronger than that obtained from the breakthrough and isotherm results for the resin, possibly due to the reduced accessibility of the immobilized ligand on resin surface. Overall, this work can facilitate future Protein A ligand design and affinity chromatography process optimization for biomanufacturing.

Role of harvest depth filtration in controlling product-related impurities for a bispecific antibody.

Background: Bispecific antibodies (BsAb) belong to a novel antibody category with advantages over traditional mono-specific therapeutic antibodies. However, product variants are also commonly seen during the production of BsAb, which poses significant challenges to downstream processing. In this study, the adsorptive characteristics of a BsAb product and its variants were investigated for a set of depth filters during primary recovery of the cell culture fluid. Methods: The retention of the BsAb product and its variants on a set of Millistak+® D0HC and X0HC depth filters were first investigated, followed by studying the mechanism of their adsorption on the depth filters. The chemical and structural properties of depth filters along with the molecular properties of the product and its variants were studied subsequently. Results: The X0HC filter was found to be able to retain a significant amount of low molecular weight (LMW) variants along with a low amount of main product retained. Different levels of retention, observed for these variants, were correlated to their different hydrophobic and charge characteristics in relation with the adsorptive properties of the depth filters used. Electrostatic, hydrophobic, and hydrogen bonding interactions were found to be the key forces to keep product variants retained on the depth filter where the higher hydrophobicity of the LMW variants may cause them to be preferentially retained. Conclusion: Harvest depth filters potentially can be utilized for retaining the BsAb variants, which depends on relative molecular properties of the product and its variants and adsorptive properties of the depth filters used.

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.

Assessing detergent-mediated virus inactivation, protein stability, and impurity clearance in biologics downstream processes.

Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance in mammalian processes, in particular for next-generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent Triton X-100 with eco-friendly alternatives. However, given Triton X-100 has been the gold standard for VI due its minimal impact on protein stability and high inactivation efficacy, inactivation by other eco-friendly detergents and its impact on protein stability is not well understood. In this study, the sugar-based detergent commonly used in membrane protein purification, n-dodecyl-ß- d-maltoside was found to be a promising alternative for VI. We investigated a panel of detergents to compare the relative VI efficacy, impact on therapeutic quality attributes, and clearance of the VI agent and other impurities through subsequent chromatographic steps. Detergent-mediated inactivation and protein stability showed comparable trends to low pH inactivation. Using experimental and modeling data, we found detergent-mediated product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Detergent-mediated aggregation was also impacted by an initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity, and critical micelle concentration. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize, and optimize conditions (concentration and duration of inactivation) for screening of detergent-mediated VI.

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes.

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.

Behavior of weakly adsorbing protein impurities in flow-through ion-exchange chromatography.

Flow-through ion-exchange chromatography is frequently used in polishing biotherapeutics, but the factors that contribute to impurity persistence are incompletely understood. A large number of dilute impurities may be encountered that exhibit physicochemical diversity, making the flow-through separation performance highly sensitive to process conditions. The analysis presented in this work develops two novel correlations that offer transferable insights into the chromatographic behavior of weakly adsorbing impurities. The first, based on column simulations and validated experimentally, delineates the relative contributions of thermodynamic, transport, and geometric properties in dictating the initial breakthrough volumes of dilute species. The Graetz number for mass transfer was found to generalize the transport contributions, enabling estimation of a threshold in the equilibrium constant below which impurity persistence is expected. Impurity adsorption equilibria are needed to use this correlation, but such data are not typically available. The second relationship presented in this work may be used to reduce the experimental burden of estimating adsorption equilibria as a function of ionic strength. A correlation between stoichiometric displacement model parameters was found by consolidating isocratic retention data for over 200 protein-pH-resin combinations from the extant literature. Coupled with Yamamoto's analysis of linear gradient elution data, this correlation may be used to estimate retentivity approximately from a single experimental measurement, which could prove useful in predicting host-cell protein chromatographic behavior.

Virus inactivation at moderately low pH varies with virus and buffer properties.

BACKGROUND: Virus inactivation is a critical operation in therapeutic protein manufacturing. Low pH buffers are a widely used strategy to ensure robust enveloped virus clearance. However, the choice of model virus can give varying results in viral clearance studies. Pseudorabies virus (SuHV) or herpes simplex virus-1 (HSV-1) are frequently chosen as model viruses to demonstrate the inactivation for the herpes family. RESULTS: In this study, SuHV, HSV-1, and equine arteritis virus (EAV) were used to compare the inactivation susceptibility at pH 4.0 and 4°C. SuHV and HSV-1 are from the same family, and EAV was chosen as a small, enveloped virus. Glycine, acetate, and citrate buffers at pH 4.0 and varying buffer strengths were studied. The inactivation susceptibility was found to be in the order of SuHV > HSV > EAV. The buffer effectiveness was found to be in the order of citrate > acetate > glycine. The smaller virus, EAV, remained stable and infectious in all the buffer types and compositions studied. CONCLUSION: The variation in inactivation susceptibility of herpes viruses indicated that SuHV and HSV cannot be interchangeably used as a virus model for inactivation studies. Smaller viruses might remain adventitiously infective at moderately low pH.

Surrogate model to screen for inactivation-based clearance of enveloped viruses during biotherapeutics process development.

Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of a minimum of 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2 to 3 days vs. 4 to 7 months.

Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic's efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

Affinity precipitation of monoclonal antibodies using ELP-Z in the elution without resolubilization mode.

This paper describes a simplified affinity precipitation process for the purification of mAbs from complex mixtures using elastin-like polypeptide fused to a single Z domain of protein A (ELP-Z). This approach eliminates several steps in the original process by directly extracting the mAb from the affinity precipitate, without the need for resolubilization. The efficacy of this elution without resolubilization (EWR) approach for obtaining pure mAb is demonstrated and the effects of mixing are examined. This simplification of the affinity precipitation process may facilitate the implementation of ELP-Z based mAb bioprocessing, particularly in a continuous scenario.

Fast and high-resolution purification of a PEGylated protein using a z2 laterally-fed membrane chromatography device.

PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In this study, we discuss the application of a newly developed z2 laterally-fed membrane chromatography (or z2LFMC) device for carrying out high-resolution purification of a PEGylated protein drug. The device used in the current study contained a stack of anion exchange (Q) membranes. The membrane bed-height of this z2LFMC device being small, it could be operated at very high flow rates, at relatively low back pressures. The primary goal was to speedily and efficiently separate a mono-PEGylated protein from impurities present in the PEGylation reaction mixture. A resin-based anion exchange column having the same ligand and bed-volume was used as the control device. The purification performance of the z2LFMC device and the control column were compared terms of resolution, recovery and purity. The z2LFMC device outperformed the control column in terms of every metric compared in this study. Higher purity (85.4% as opposed to 77.9%) and higher recovery (28% greater) of the target mono-PEGylated protein were obtained using the z2LFMC device at 20-time higher speed. These results clearly demonstrate that the z2LFMC device could be a faster and more efficient alternative to resin-based columns for purification of biopharmaceuticals.

Physiochemical properties of enveloped viruses and arginine dictate inactivation.

BACKGROUND: Therapeutic protein manufacturing would benefit by having an arsenal of ways to inactivate viruses. There have been many publications on the virus inactivation ability of arginine at pH 4.0, but the mechanism of this inactivation is unknown. This study explored how virus structure and solution conditions enhance virus inactivation by arginine and leads to a better understanding of the mechanism of virus inactivation by arginine. RESULTS: Large diameter viruses from the Herpesviridae family (SuHV-1, HSV-1) with loosely packed lipids were highly inactivated by arginine, whereas small diameter, enveloped viruses (equine arteritis virus (EAV) and bovine viral diarrhea virus (BVDV)) with tightly packed lipids were negligibly inactivated by arginine. To increase the inactivation of viruses resistant to arginine, arginine-derivatives and arginine peptides were tested. Derivates and peptides demonstrated that a greater capacity for clustering and added hydrophobicity enhanced virus inactivation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) detected increases in virus size after arginine exposure, supporting the mechanism of lipid expansion. CONCLUSIONS: Arginine most likely interacts with the lipid membrane to cause inactivation. This is shown by larger viruses being more sensitive to inactivation and expansion of the viral size. The enhancement of arginine inactivation when increased hydrophobic molecules are present or arginine is clustered demonstrates a potential mechanism of how arginine interacts with the lipid membrane.