Understanding the Impact of Combined Hydrodynamic Shear and Interfacial Dilatational Stress, on Interface-Mediated Particle Formation for Monoclonal Antibody Formulations.

During biomanufacturing, several unit operations expose solutions of biologics to multiple stresses, such as hydrodynamic shear forces due to fluid flow and interfacial dilatational stresses due to mechanical agitation or bubble collapse. When these stresses individually act on proteins adsorbed to interfaces, it results in an increase in protein particles in the bulk solution, a phenomenon referred to as interface-induced protein particle formation. However, an understanding of the dominant cause, when multiple stresses are acting simultaneously or sequentially, on interface-induced protein particle formation is limited. In this work, we established a unique set-up using a peristaltic pump and a Langmuir-Pockels trough to study the impact of hydrodynamic shear stress due to pumping and interfacial dilatational stress, on protein particle formation. Our experimental results together demonstrate that for protein solutions subjected to various combinations of stress (i.e., interfacial and hydrodynamic stress in different sequences), surface pressure values during adsorption and when subjected to compression/dilatational stresses, showed no change, suggesting that the interfacial properties of the protein film are not impacted by pumping. The concentration of protein particles is an order of magnitude higher when interfacial dilatational stress is applied at the air-liquid interface, compared to solutions that are only subjected to pumping. Furthermore, the order in which these stresses are applied, have a significant impact on the concentration of protein particles measured in the bulk solution. Together, these studies conclude that for biologics exposed to multiple stresses throughout bioprocessing and manufacturing, exposure to air-liquid interfacial dilatational stress is the predominant mechanism impacting protein particle formation at the interface and in the bulk solution.

The role of intermolecular interactions on monoclonal antibody filtration through virus removal membranes.

The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through three different commercial virus filters (Viresolve Pro, Viresolve NFP, and Pegasus SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100-fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering (DLS). The mAb with the lowest filtrate flux for all three membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography (HIC) column. These results strongly suggest that the flux behavior is dominated by reversible self-association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs/solution conditions.

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.

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.

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.

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.

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.

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.

Using continuous chromatography methodology to achieve high-productivity and high-purity enrichment of charge variants for analytical characterization.

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants.

Process analytical technology for on-line monitoring of quality attributes during single-use ultrafiltration/diafiltration.

Process analytical technology (PAT) is a fast-growing field within bioprocessing that enables innovation in biological drug manufacturing. This study demonstrates novel PAT methods for monitoring multiple quality attributes simultaneously during the ultrafiltration and diafiltration (UF/DF) process operation, the final step of monoclonal antibody (mAb) purification. Size exclusion chromatography (SEC) methods were developed to measure excipients arginine, histidine, and high molecular weight (HMW) species using a liquid chromatography (LC) system with autosampler for both on-line and at-line PAT modes. The methods were applied in UF/DF studies for the comparison of single-use tangential flow filtration (TFF) cassettes to standard reusable cassettes to achieve very high concentration mAb drug substance (DS) in the order of 100-200 g/L. These case studies demonstrated that single-use TFF cassettes are a functionally equivalent, low-cost alternative to standard reusable cassettes, and that the on-line PAT measurement of purity and excipient concentration was comparable to orthogonal offline methods. These PAT applications using an on-line LC system equipped with onboard sample dilution can become a platform system for monitoring of multiple attributes over a wide dynamic range, a potentially valuable tool for biological drug development and manufacturing.

Control of leached beta-glucan levels from depth filters by an improved depth filtration flush strategy.

Beta-glucans are polysaccharides of D-glucose monomers linked by (1-3) beta-glycosidic bonds, are found to have a potential immunogenicity risk in biotherapeutic products, and are labeled as process contaminants. A common source of beta-glucans is from the cellulose found in traditional depth filter media. Typically, beta-glucan impurities that leach into the product from the primary clarification depth filters can be removed by the subsequent bind-and-elute affinity chromatography capture step. Beta-glucans can also be removed by a bind-and-elute cation exchange chromatography step, which is useful for removing beta-glucans introduced by a post-Protein A depth filtration step. However, the increasing prevalence of flowthrough polishing chromatography poses a challenge for beta-glucan removal due to the lack of any bind-and-elute chromatography steps after the post-Protein A depth filter. In this work, a depth filter flush strategy was developed to control beta-glucan leaching into the product pool. Different loading conditions for the depth filtration and subsequent chromatography steps were evaluated to determine the robustness of the optimized flush strategy. Carry through runs demonstrated greater than two-fold reduction in beta-glucan levels using the optimized wash as compared to standard filter flush conditions.

Real-time monitoring of quality attributes by in-line Fourier transform infrared spectroscopic sensors at ultrafiltration and diafiltration of bioprocess.

Technologies capable of monitoring product quality attributes and process parameters in real time are becoming popular due to the endorsement of regulatory agencies and also to support the agile development of biotherapeutic pipelines. The utility of vibrational spectroscopic techniques such as Fourier transform mid-infrared (Mid-IR) and multivariate data analysis (MVDA) models allows the prediction of multiple critical attributes simultaneously in real time. This study reports the use of Mid-IR and MVDA model sensors for monitoring of multiple attributes (excipients and protein concentrations) in real time (measurement frequency of every 40 s) at ultrafiltration and diafiltration (UF/DF) unit operation of biologics manufacturing. The platform features integration of fiber optic Mid-IR probe sensors to UF/DF set up at the bulk solution and through a flow cell at the retentate line followed by automated Mid-IR data piping into a process monitoring software platform with pre-loaded partial least square regression (PLS) chemometric models. Data visualization infrastructure is also built-in to the platform so that upon automated PLS prediction of excipients and protein concentrations, the results were projected in a graphical or numerical format in real time. The Mid-IR predicted concentrations of excipients and protein show excellent correlation with the offline measurements by traditional analytical methods. Absolute percent difference values between Mid-IR predicted results and offline reference assay results were ≤5% across all the excipients and the protein of interest; which shows a great promise as a reliable process analytical technology tool.

Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review.

To achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold-up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self-association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity-lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high-concentration drug substance manufacturing, such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high-concentration drug substance manufacturing.

Continuous In-Line Virus Inactivation for Next Generation Bioprocessing.

Viral inactivation plays a critical role in assuring the safety of monoclonal antibody (mAb) therapeutics. Traditional viral inactivation involves large holding tanks in which product is maintained at a target low pH for a defined hold time, typically 30-60 min. The drive toward continuous processing and improved facility utilization has provided motivation for development of a continuous viral inactivation process. To this end, a lab-scale prototype viral inactivation system was designed, built, and characterized. Multiple incubation chamber designs are evaluated to identify the optimal design that enables narrow residence time distributions in continuous flow systems. Extensive analysis is conducted supporting rapid low pH viral inactivation and included evaluations with multiple viruses, a range of pH levels, buffer compositions, mAb concentrations, and temperatures. Multiple test conditions are evaluated using the in-line system and results compared to traditional batch-mode viral inactivation. Comparability in kinetics of virus inactivation suggests equivalency between the two approaches.

Effects of urea on selectivity and protein-ligand interactions in multimodal cation exchange chromatography.

Nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were employed in concert with chromatography to provide insight into the effect of urea on protein-ligand interactions in multimodal (MM) chromatography. Chromatographic experiments with a protein library in ion exchange (IEX) and MM systems indicated that, while urea had a significant effect on protein retention and selectivity for a range of proteins in MM systems, the effects were much less pronounced in IEX. NMR titration experiments carried out with a multimodal ligand, and isotopically enriched human ubiquitin indicated that, while the ligand binding face of ubiquitin remained largely intact in the presence of urea, the strength of binding was decreased. MD simulations were carried out to provide further insight into the effect of urea on MM ligand binding. These results indicated that, while the overall ligand binding face of ubiquitin remained the same, there was a reduction in the occupancy of the MM ligand interaction region along with subtle changes in the residues involved in these interactions. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems and also provides an in-depth analysis of how MM ligand-protein interactions are altered in the presence of this fluid phase modifier.

Improving selectivity in multimodal chromatography using controlled pH gradient elution.

Externally generated pH gradients are employed on a multimodal cation exchange chromatographic resin to improve the selectivity for a mixture of model proteins. By combining controlled pH gradients with the unique selectivities arising from the multiple interaction types exhibited by the multimodal resin, the separation of the protein mixture is significantly improved as compared to linear salt gradient operation. Several gradient conditions are explored and a shallow gradient from pH 3.8 to 5.5 is shown to be able to resolve the proteins. This work provides proof of concept for the use of pH gradients in multimodal chromatography and sets the stage for future applications.

Probing multimodal ligand binding regions on ubiquitin using nuclear magnetic resonance, chromatography, and molecular dynamics simulations.

Site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation experiments, and molecular dynamics (MD) simulations are employed in concert with chromatographic experiments to provide insight into protein-ligand interactions in multimodal chromatographic systems. In previous studies, a preferred binding region was identified on the surface of the protein ubiquitin for binding with a multimodal ligand. In this study, site-directed mutagenesis is used to enable direct NMR evaluation of the mutant protein as compared to the wild type. It is found that reversing the charge of a key residue (K6E) in the proposed preferred binding region results in substantial decreases in the magnitude of the ligand-induced NMR chemical shift perturbations relative to those detected for the wild type protein, particularly for residues located within the preferred binding region. These NMR results also indicate a decrease in ligand affinity, consistent with the weaker chromatographic retention observed for the mutant as compared to the wild type on a multimodal cation exchange resin. MD simulation results provide additional insight at a molecular level and demonstrate that many residues located within the preferred binding region exhibit weaker binding interactions due to the mutation. The analysis suggests that multimodal ligand binding consists of initial localization of the ligand by long-ranged electrostatic interactions followed by multiple short-ranged synergistic interactions to attain high affinities of the ligand to specific residues.

Mobile phase modifier effects in multimodal cation exchange chromatography.

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.

Detection of trace proteins in multicomponent mixtures using displacement chromatography.

Model protein feed mixtures containing three abundant and seven trace proteins at various concentrations were identified and employed in a series of displacement experiments. Reversed-phase liquid chromatography (RPLC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry were used to evaluate the compositions of both the feed mixtures and effluent fractions from the displacement experiments. The results demonstrated that trace proteins were focused at the boundaries between the abundant solutes where they were enriched and concentrated. For many of the multicomponent feed mixtures, mass spectrometry analyses of the displacement column effluent fractions resulted in the identification of trace proteins that were not detectable in the feed. In addition, the use of minimal or no salt in the carrier solutions enabled the analysis of displacement fractions by direct infusion mass spectrometry. These results are significant in that they indicate that while the presence of abundant proteins can often be problematic for the detection of trace components, displacement chromatography may be able to employ these abundant proteins to focus trace proteins in the displacement train, thus facilitating detection.