Optical Coherence Tomography Velocimetry for In-Line Processing of Biologics: Concentrated and Gelling Monoclonal Antibody Solutions.

Viscosity Behavior of High-Concentration Monoclonal Antibody Solutions: Correlation with Interaction Parameter and Electroviscous Effects

Evaluating the Role of the Air-Solution Interface on the Mechanism of Subvisible Particle Formation Caused by Mechanical Agitation for an IgG1 mAb

Ongoing efforts in the biopharmaceutical industry to enhance productivity and reduce manufacturing costs include development of intensified, linked, and/or continuous processes. One approach to improve productivity and process economics of the polishing step (i.e., anion exchange chromatography) is to preconcentrate the product intermediate using a single-pass tangential flow filtration step before loading on the resin. This intensification of the polishing step consequently leads to changes in product intermediate concentration for subsequent virus filtration operations, potentially impacting filter performance and methods for evaluating viral clearance. The filtrate flux performance of a virus filtration operation was evaluated with monoclonal antibody (mAb) solutions of varying concentrations. These data were used to evaluate the effect on filter sizing for a hypothetical mAb perfusion process. The optimum mAb concentration to minimize the area of the virus filter was a function of the filtration step duration and reflected the competing effects of increasing concentration and decreasing volumetric flux on the membrane productivity. mAb solutions at high and low concentrations were used to evaluate viral clearance with extended filtration times (e.g., 24-72 h) simulating continuous processing conditions. Modifications to more traditional filtration viral clearance study methods were required to avoid experimental artifacts associated with the extended filtration time. No virus passage through the filter was observed under these conditions, similar to previous results for batch processes. These data demonstrate the feasibility of obtaining effective virus removal even when mAb concentration and filtrations times are increased by up to an order of magnitude from current common practices.

To investigate relationship between second virial coefficient B (2) and viscosity and aggregation propensity of highly concentrated monoclonal antibody (MAbs) solutions. Intermolecular interactions of 3 MAbs solutions with varying pH were characterized according to B (2) estimated by analytical ultracentrifugation sedimentation equilibrium with initial loading concentrations <10mg/mL. Viscosity measurements and stability assessments of MAb solutions at concentrations higher than 100mg/mL were conducted. B (2) of all MAb solutions depended on solution pH and have qualitative correlation with viscosity and aggregation propensity. The more negative the B (2) values, the more viscous the solution, acquiring increased propensity to aggregate. Solutions with B (2) values of ~2 × 10(-5)mL·mol/g(2) acquire similar viscosity and aggregation propensity regardless of amino acid sequences; for solutions with negative B (2) values, viscosity and aggregation propensity differed depending on sequences. Results suggest attractive intermolecular interactions represented by negative B (2) values are influenced by surface properties of individual MAbs. B (2) can be used, within certain limitations, as an effective indicator of viscosity and aggregation propensity of highly concentrated MAb solutions.

Weak Interactions Govern the Viscosity of Concentrated Antibody Solutions: High-Throughput Analysis Using the Diffusion Interaction Parameter

PurposeThis study addresses the effect of freezing and thawing on a therapeutic monoclonal antibody (mAb) solution and the corresponding buffer formulation. Particle formation, crystallization behaviour, morphology changes and cryo-concentration effects were studied after varying the freezing and thawing rates, buffer formulation and protein concentration. The impact of undergoing multiple freeze/thaw (FT)-cycles at controlled and uncontrolled temperature rates on mAb solutions was investigated in terms of particle formation.MethodsPhysicochemical characteristics were analysed by Differential Scanning Calorimetry whereas morphology changes are visualized by cryomicroscopy measurements. Micro Flow Imaging, Archimedes and Dynamic Light Scattering were used to investigate particle formation.ResultsData retrieved in the present study emphasizes the damage caused by multiple FT-cyles and the need for sucrose as a cryoprotectant preventing cold-crystallization specifically at high protein concentrations. Low protein concentrations cause an increase of micron particle formation. Low freezing rates lead to a decreased particle number with increased particle diameter.ConclusionThe overall goal of this research is to gain a better understanding of the freezing and thawing behaviour of mAb solutions with the ultimate aim to optimize this process step by reducing the unwanted particle formation, which also includes protein aggregates.

Viscosity increase/gelation of therapeutic IgG monoclonal antibodies induced by Zn2+: One possible root cause of clogging of staked-in-needle prefilled syringes

Understanding the Freezing of Biopharmaceuticals: First-Principle Modeling of the Process and Evaluation of Its Effect on Product Quality

Understanding protein-protein interactions and formation of reversible oligomers (clusters) in concentrated monoclonal antibody (mAb) solutions is necessary for designing stable, low viscosity (η) concentrated formulations for processing and subcutaneous injection. Here we characterize the strength (K) of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors (Seff(q)) from small-angle X-ray scattering (SAXS) using coarse-grained molecular dynamics simulations. Best fit simulations additionally provide cluster size distributions, fractal dimensions, cluster occluded volume, and mAb coordination numbers. These equilibrium properties are utilized in a model to account for increases in viscosity caused by occluded volume in the clusters (packing effects) and dissipation of stress across lubricated fractal clusters. Seff(q) is highly sensitive to K at 75 mg/mL where mAbs can mutually align to form SRA contacts but becomes less sensitive at 200 mg/mL as steric repulsion due to packing becomes dominant. In contrast, η at 200 mg/mL is highly sensitive to SRA and the average cluster size from SAXS/simulation, which is observed to track the cluster relaxation time from shear thinning. By analyzing the distribution of sub-bead hot spots on the 3D mAb surface, we identify a strongly attractive hydrophobic patch in the complementarity determining region (CDR) at pH 4.5 that contributes to the high K and consequently large cluster sizes and high η. Adding NaCl screens electrostatic interactions and increases the impact of hydrophobic attraction on cluster size and raises η, whereas nonspecific binding of Arg attenuates all SRA, reducing η. The hydrophobic patch is absent at higher pH values, leading to smaller K, smaller clusters, and lower η. This work constitutes a first attempt to use SAXS and CG modeling to link both structural and rheological properties of concentrated mAb solutions to the energetics of specific hydrophobic patches on mAb surfaces. As such, our work opens an avenue for future research, including the possibility of designing coarse-grained models with physically meaningful interacting hot spots.

Parallel plate and microstrip resonator experiments show that NbN films have large magnetic penetration depth and a peak RF critical current density which either matches or exceeds the DC critical current density, depending on the temperature. The large penetration depth, /spl lambda/, suggests that narrow NbN transmission lines will have increased inductive nonlinearities, when compared with other superconductors. The relation between the DC and peak RF critical current densities indicates that by modeling the RF current distribution a simple DC breakdown measurement can be used to estimate a transmission line's RF current carrying capacity.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

To discover, elucidate the structure-activity relationship (SAR), and explore the mechanism of action of excipients able to drastically lower the viscosities of concentrated aqueous solutions of humanized monoclonal antibodies (MAbs). Salts prepared from hydrophobic cations and anions were dissolved into humanized MAbs solutions. Viscosities of the resulting solutions were measured as a function of the nature and concentration of the salts and MAbs. Even at moderate concentrations, some of the salts prepared herein were found to reduce over 10-fold the viscosities of concentrated aqueous solutions of several MAbs at room temperature. To be potent viscosity-lowering excipients, the ionic constituents of the salts must be hydrophobic, bulky, and aliphatic. A mechanistic hypothesis explaining the observed salt effects on MAb solutions' viscosities was proposed and verified.

Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

To further advance a subcutaneous injection of monoclonal antibodies (mAbs) at elevated concentrations, novel concepts are needed to lower the viscosity. The addition of high concentrations of cosolutes, namely, arginine glutamate (Arg·Glu) or Arg·HCl, reduced the viscosity of a ∼250 mg/mL mAb solution up to 6-fold. With Arg·Glu, the viscosity of the mAb solution was reduced to 30 cP and for a polyclonal sheep IgG solution to 17 cP both at ∼250 mg/mL. Viscosities went through a maximum at the mAb isoelectric point for solutions with Arg·Glu or Arg·HCl. In contrast the viscosity was only weakly affected by NaCl or the preferentially excluded molecule trehalose. The large viscosity reduction from Arg may be attributed to direct binding to the mAb, resulting in suppression of both hydrophobic and local anisotropic electrostatic attraction. Aggregate formation was negligible for high cosolute mAb solutions as demonstrated by SEC even after 8 weeks of 25 °C storage.

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.

To achieve uniform thickness of coated films using non-Newtonian fluids, we are often required well-designed geometries of flow channel in a slot die. This is the case especially for fluids with the properties of both shear thinning and yield stress which is non-negligible in a range of low shear rate such as in an intermittent die coating. In the present work, flow in the slot die has been studied using Herschel-Bulkley fluid expressing both shear thinning and yield stress. The Herschel-Bulkley fluid includes power-law and Bingham fluid models comprehensively. This fluid model is applicable to many real fluids. We derive simplified equations for the outflow distribution as functions of power-law index, yield stress and the geometry parameters of the die. Furthermore, we propose a useful designing method for predicting the optimum geometry so as to guarantee uniformity of outflow from the die slot, based on the Herschel-Bulkley fluid. The value of this method is confirmed by comparing it with experimental results and with previous theoretical results for the power-law and Bingham fluid models.