Continuous precipitation-filtration process for initial capture of a monoclonal antibody product using a four-stage countercurrent hollow fiber membrane washing step.

The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using precipitation as a low-cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. In this work, a commercially relevant mAb was purified from clarified cell culture fluid using a tubular flow precipitation reactor with dewatering and washing provided by tangential flow microfiltration. The particle morphology was evaluated using an inline high-resolution optical probe, providing quantitative data on the particle size distribution throughout the precipitation process. Data were obtained in both a lab-built 2-stage countercurrent washing system and a commercial countercurrent contacting skid that provided 4 stages of continuous washing. The processes were operated continuously for 2 h with overall mAb yield of 92 ± 3% and DNA removal of nearly 3 logs in the 4-stage system. The high DNA clearance was achieved by selective redissolution of the mAb using a low pH acetate buffer. Host cell protein clearance was 0.59 ± 0.08 logs, comparable to that based on model predictions. The process mass intensity was slightly better than typical Protein A processes and could be significantly improved by preconcentration of the antibody feed material.

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing.

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.

Surfactant-induced Marangoni transport of lipids and therapeutics within the lung.

Understanding the fundamentals of surface transport on thin viscous films has important application in pulmonary drug delivery. The human lung contains a large-area interface between its complex fluid lining and inhaled air. Marangoni flows driven by surface tension gradients along this interface would promote enhanced distribution of inhaled therapeutics by carrying them from where they are deposited in the upper airways, along the fluid interface to deeper regions of the lung. Motivated by the potential to improve therapies for acute and chronic lung diseases, we review recent progress in modeling and experimental studies of Marangoni transport induced by the deposition of surfactant-containing microliter drops and liquid aerosols (picoliter drops) onto a fluid interface. The roles of key system variables are identified, including surfactant solubility, drop miscibility with the subphase, and the thickness, composition and surface properties of the subphase liquid. Of particular interest is the unanticipated but crucial role of aerosol processing to achieve Marangoni transport via phospholipid vesicle dispersions, which are likely candidates for a biocompatible delivery system. Progress in this field has the potential to not only improve outcomes in patients with chronic and acute lung diseases, but also to further our understanding of surface transport in complex systems.

Moringa oleifera Seed Protein Adsorption to Silica: Effects of Water Hardness, Fractionation, and Fatty Acid Extraction.

Motivated by the proposed use of cationic protein-modified sand for water filtration in developing nations, this study concerns the adsorption of Moringa oleifera seed proteins to silica surfaces. These proteins were prepared in model waters of varying hardness and underwent different levels of fractionation, including fatty acid extraction and cation exchange chromatography. Adsorption isotherms were measured by ellipsometry, and the zeta potentials of the resulting protein-decorated surfaces were measured by the rotating disk streaming potential method. The results indicate that the presence of fatty acids has little effect on the M. oleifera cationic protein adsorption isotherm. Adsorption from the unfractionated extract was indistinguishable from that of the cationic protein isolates at low concentrations but yielded significantly greater extents of adsorption at high concentrations. Adsorption isotherms for samples prepared in model hard and soft fresh waters were indistinguishable from each other over the measured bulk solution concentration range, but adsorption from hard or soft water was more extensive than adsorption from deionized water at moderate protein concentrations. Streaming potential measurements showed that adsorption reversed the net sign of the zeta potential of silica from negative to positive for all protein fractions and water hardness conditions at protein bulk concentrations as low as 0.03 µg/mL. This suggests that sands can be effectively modified with M. oleifera proteins using small amounts of seed extract under various local water hardness conditions. Finally, ellipsometry indicated that M. oleifera proteins adsorb irreversibly with respect to rinsing in these model fresh waters, suggesting that the modified sand would be stable on repeated use for water filtration. These studies may aid in the design of a simple, effective, and sustainable water purification device for developing nations.

Evolution and Disappearance of Solvent Drops on Miscible Polymer Subphases.

Traditionally, an interface is defined as a boundary between immiscible phases. However, previous work has shown that even when two fluids are completely miscible, they maintain a detectable "effective interface" for long times. Miscible interfaces have been studied in various systems of two fluids with a single boundary between them. However, this work has not extended to the three-phase system of a fluid droplet placed on top of a miscible pool. We show that these three-phase systems obey the same wetting conditions as immiscible systems, and that their drop shapes obey the Augmented Young-Laplace Equation. Over time, the miscible interface diffuses and the shape of the drop evolves. We place 2-microliter drops of water atop miscible poly(acrylamide) solutions. The drop is completely wetted by the subphase, and then remains detectable beneath the surface for many minutes. An initial effective interfacial tension can be approximated to be on the order of 0.5 mN/m using the capillary number. Water and poly(acrylamide) are completely miscible in all concentrations, and yet, when viewed from the side, the drop maintains a capillary shape. Study of this behavior is important to the understanding of effective interfaces between miscible polymer phases, which are pervasive in nature.

Transport of a partially wetted particle at the liquid/vapor interface under the influence of an externally imposed surfactant generated Marangoni stress.

Marangoni flows offer an interesting and useful means to transport particles at fluid interfaces with potential applications such as dry powder pulmonary drug delivery. In this article, we investigate the transport of partially wetted particles at a liquid/vapor interface under the influence of Marangoni flows driven by gradients in the surface excess concentration of surfactants. We deposit a microliter drop of soluble (sodium dodecyl sulfate aqueous solution) surfactant solution or pure insoluble liquid (oleic acid) surfactant on a water subphase and observe the transport of a pre-deposited particle. Following the previous observation by Wang et al. [1] that a surfactant front rapidly advances ahead of the deposited drop contact line initiates particle motion but then moves beyond the particle, we now characterize the two dominant, time- and position-dependent forces acting on the moving particle: 1) a surface tension force acting on the three-phase contact line around the particle periphery due to the surface tension gradient at the liquid/vapor interface which always accelerates the particle and 2) a viscous force acting on the immersed surface area of the particle which accelerates or decelerates the particle depending on the difference in the velocities of the liquid and particle. We find that the particle velocity evolves over time in two regimes. In the acceleration regime, the net force on the particle acts in the direction of particle motion, and the particle quickly accelerates and reaches a maximum velocity. In the deceleration regime, the net force on the particle reverses and the particle decelerates gradually and stops. We identify the parameters that affect the two forces acting on the particle, including the initial particle position relative to the surfactant drop, particle diameter, particle wettability, subphase thickness, and surfactant solubility. We systematically vary these parameters and probe the spatial and temporal evolution of the two forces acting on the particle as it moves along its trajectory in both regimes. We find that a larger particle always lags behind the smaller particle when placed at an equal initial distance from the drop. Similarly, particles more deeply engulfed in the subphase lag behind those less deeply engulfed. Further, the extent of particle transport is reduced as the subphase thickness decreases, due to the larger velocity gradients in the subphase recirculation flows.

Autophobing on liquid subphases driven by the interfacial transport of amphiphilic molecules.

We investigated the phenomenon of incomplete wetting of a high-energy liquid subphase by drops of pure amphiphilic molecules as well as drops of amphiphile solutions that are immiscible with the subphase. We show that amphiphiles escape across the contact line of the drop, move on the subphase/vapor interface, and form a submonolayer or full monolayer external to the drop. If this monolayer is sufficiently dense, then it can reduce the surface tension of the subphase, raise the contact angle of the drop, and prevent the drop from fully wetting the subphase. This phenomenon is called autophobing and has been extensively studied on solid substrates. For the liquid subphase studied here, we measure the surface tensions of the three relevant interfaces before and after the drop is deposited. The measured surface tension external to the drop shows that amphiphiles can move across the contact line and form a monolayer outside of the drop. In some cases, at equilibrium, the monolayer is in a sufficiently packed state to create the nonwetting condition. In other cases, at equilibrium the monolayer density is insufficient to lower the surface tension enough to achieve the nonwetting condition. Unlike on solid substrates where the formation of the monolayer external to the drop is kinetically hindered, the amphiphiles can move rapidly across the liquid subphase by Marangoni-driven surface transport, and local equilibrium is achieved. However, because the amphiphile inventory and subphase area are limited, the achievement of autophobing on a liquid subphase depends not only on the instrinsic subphase/amphiphile interaction but also on the total amphiphile inventory and area of the liquid subphase.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body's innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte's Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

The conformation of the poly(ethylene glycol) chain in mono-PEGylated lysozyme and mono-PEGylated human growth hormone.

Covalent conjugation of poly(ethylene glycol) or "PEGylation" has proven an effective strategy to improve pharmaceutical protein efficacy by hindering recognition by proteases, inhibitors, and antibodies and by retarding renal clearance. Because it determines the strength and range of intermolecular steric forces and the hydrodynamic properties of the conjugates, the configuration of protein-conjugated PEG chains is the key factor determining how PEGylation alters protein in vivo circulation time. Mono-PEGylated proteins are typically described as having a protective PEG shroud wrapped around the protein, but recent dynamic light scattering studies suggested that conjugates adopt a dumbbell configuration, with a relatively unperturbed PEG random coil adjacent to the globular protein. We used small-angle neutron scattering (SANS) to distinguish between the dumbbell model and the shroud model for chicken-egg lysozyme and human growth hormone covalently conjugated to a single 20 kDa PEG chain. The SANS contrast variation technique was used to isolate the PEG portion of the conjugate. Scattering intensity profiles were well described by the dumbbell model and inconsistent with the shroud model.

Surface tension gradient driven spreading on aqueous mucin solutions: a possible route to enhanced pulmonary drug delivery.

Surface tension gradient driven, or "Marangoni", flow can be used to move exogenous fluid, either surfactant dispersions or drug carrying formulations, through the lung. In this paper, we investigate the spreading of aqueous solutions of water-soluble surfactants over entangled, aqueous mucin solutions that mimic the airway surface liquid of the lung. We measure the movement of the formulation by incorporating dyes into the formulation while we measure surface flows of the mucin solution subphase using tracer particles. Surface tension forces and/or Marangoni stresses initiate a convective spreading flow over this rheologically complex subphase. As expected, when the concentration of surfactant is reduced until its surface tension is above that of the mucin solution, the convective spreading does not occur. The convective spreading front moves ahead of the drop containing the formulation. Convective spreading ends with the solution confined to a well-defined static area which must be governed by a surface tension balance. Further motion of the spread solution progresses by much slower diffusive processes. Spreading behaviors are qualitatively similar for formulations based on anionic, cationic, or nonionic surfactants, containing either hydrophilic or hydrophobic dyes, on mucin as well as on other entangled aqueous polymer solution subphases. This independence of qualitative spreading behaviors from the chemistry of the surfactant and subphase indicates that there is little chemical interaction between the formulation and the subphase during the spreading process. The spreading and final solution distributions are controlled by capillary and hydrodynamic phenomena and not by specific chemical interactions among the components of the system. It is suggested that capillary forces and Marangoni flows driven by soluble surfactants may thereby enhance the uniformity of drug delivery to diseased lungs.

Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface.

Covalent grafting of poly(ethylene glycol) chains to proteins ("PEGylation") is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surface-induced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface.

High throughput solubility and redissolution screening for antibody purification via combined PEG and zinc chloride precipitation.

As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2 ) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7-7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.

Flow regime transitions and effects on solute transport in surfactant-driven Marangoni flows.

HYPOTHESIS: Surfactant-driven Marangoni flow on liquid films is predicted to depend on subphase depth and initial surface tension difference between the subphase and deposited surfactant solution drop. Changes in flow behavior will impact transport of soluble species entrained in the Marangoni flow along the surface. In extreme cases, the subphase film may rupture, limiting transport. Understanding this behavior is important for applications in drug delivery, coatings, and oil spill remediation. EXPERIMENTS: A trans-illumination optical technique measured the subphase height profiles and drop content transport after drop deposition when varying initial subphase depth, surfactant concentration, and subphase viscosity. FINDINGS: Three distinct flow regimes were identified depending on the subphase depth and surfactant concentration and mapped onto an operating diagram. These are characterized as a "central depression" bounded by an outwardly traveling ridge, an "annular depression" bounded by a central dome and the traveling ridge, and an "annular dewetting" when the subphase ruptures. Well above the critical micelle concentration, transitions between regimes occur at characteristic ratios of gravitational and initial surface tension gradient stresses; transitions shift when surfactant dilution during spreading weakens the stress before the completion of the spreading event. Drop contents travel with the ridge, but dewetting hinders transport.

Aerosolizing Lipid Dispersions Enables Antibiotic Transport Across Mimics of the Lung Airway Surface Even in the Presence of Pre-existing Lipid Monolayers.

BACKGROUND: Secondary lung infections are the primary cause of morbidity associated with cystic fibrosis lung disease. Aerosolized antibiotic inhalation is potentially advantageous but has limited effectiveness due to altered airway aerodynamics and deposition patterns that limit drug access to infected regions. One potential strategy to better reach infected areas is to formulate aerosols with surfactants that induce surface tension gradients and drive postdeposition drug dispersal via Marangoni transport along the airway surface liquid (ASL). Since this relies on surfactant-induced surface tension reduction, the presence of endogenous lipid monolayers may hinder drug dispersal performance. METHODS: Tobramycin solutions were formulated with dipalmitoylphosphatidylcholine (DPPC), a major component of endogenous pulmonary surfactant, to drive postdeposition aerosol dispersal across a model ASL based on a liquid layer or "subphase" of aqueous porcine gastric mucin (PGM) solution with predeposited DPPC monolayers to mimic the endogenous surfactant. In vitro subphase samples were collected from regions outside the aerosol deposition zone and assayed for tobramycin concentration using a closed enzyme donor immunoassay. The motion of a tracking bead across the subphase surface and the corresponding decrease in surface tension on aerosol deposition were tracked both with and without a predeposited DPPC monolayer. The surface tension/area isotherm for DPPC on PGM solution subphase was measured to aid in the interpretation of the tobramycin dispersal behavior. RESULTS AND CONCLUSIONS: Transport of tobramycin away from the deposition region occurs in aerosols formulated with DPPC whether or not predeposited lipid is present, and tobramycin concentrations are similar in both cases across biologically relevant length scales (∼8 cm). When DPPC is deposited from an aerosol, it induces ultralow surface tensions (<5 mN/m), which drive Marangoni flows, even in the presence of a dense background layer of DPPC. Therefore, aerosolized phospholipids, such as DPPC, will likely be effective spreading agents in the human lung.

Aggregation of lysozyme and of poly(ethylene glycol)-modified lysozyme after adsorption to silica.

Surface-induced aggregation is a common instability during protein storage, delivery and purification. This aggregation can lead to the formation of fibrils rich in intermolecular beta-sheet structure. Techniques to probe surface-clustering are limited. Here we use protein intrinsic fluorescence and thioflavin T probe fluorescence in a total internal reflection fluorescence (TIRF) sampling geometry to simultaneously monitor the kinetics of adsorption and aggregation for chicken egg lysozyme on a silica surface. We observe a slow surface-induced aggregation process that continues well after the lysozyme adsorption kinetics have plateaued. The rate of surface-induced aggregation is independent of the lysozyme concentration in solution. Consistent with the clustering observed via thioflavin T fluorescence, infrared amide I band spectra also show a 1.5-fold increase in intermolecular beta-sheet content upon lysozyme adsorption. Tryptophan emission spectra show no evidence for any tertiary structural change upon adsorption. Furthermore, we observe that the covalent modification of lysozyme with a single poly(ethylene glycol) (PEG) grafted chain does not inhibit aggregation on the surface, but a second PEG graft significantly inhibits the intermolecular beta-sheet formation.

Effect of polyelectrolyte-surfactant complexation on Marangoni transport at a liquid-liquid interface.

Complexation of surfactants and oppositely charged polyelectrolytes is expected to alter Marangoni transport at a fluid interface compared to either single component system due to altered interfacial tension isotherms and mass transfer rates as well as adsorption irreversibility effects. We investigate Marangoni transport at the oil/water interface by passing mixtures of the anionic surfactant sodium dodecyl sulfate (SDS) and cationic polyelectrolyte poly(3-(2-methylpropionamide)propyl) trimethylammonium chloride-acrylamide (poly[AM-MAPTAC]), or rinsing solutions, over an oil/water interface in a radial, stagnation point flow. The displacements of adsorbed tracer particles are recorded through optical microscopy. The net displacement, defined as the sum of the displacements occurring during the adsorption and desorption stages of one application and rinsing cycle, is up to 10 times greater for complexing surfactant/polymer mixtures compared to either single component system. The enhanced net displacement is largely determined by the enhanced transport upon adsorption, while the reverse displacement that would normally occur upon rinsing is partially suppressed by partially irreversible polymer adsorption at the oil/water interface. In addition to effects of complexation on interfacial tension gradient induced flow, complexation effects on the bulk, and possibly interfacial, viscosity also influence the interfacial transport.

Transient Marangoni transport of colloidal particles at the liquid/liquid interface caused by surfactant convective-diffusion under radial flow.

HYPOTHESIS: Interfacial tension gradients at a liquid/liquid interface drive Marangoni flows. When colloidal particles are adsorbed to an interface in systems with spatial and temporal gradients of surfactant concentration, these interfacial flows can be potentially significant contributors to the direction and rate of particle transport. EXPERIMENTS: In this work, we use optical microscopy to measure the interfacial velocities of 5µm diameter polystyrene latex particles adsorbed at an oil/water interface, using olive oil to represent polar oils often encountered in cleaning applications. FINDINGS: On surfactant adsorption the maximum interfacial velocity scales linearly with bulk surfactant concentration, even for concentrations exceeding the critical micelle concentration (CMC). The maximum interfacial velocity weakly decreases with increasing flow rate, but it varies non-monotonically with the radial distance from the inlet. Upon surfactant desorption into a rinse solution, the maximum velocity increases with increasing concentration of the original surfactant solution, but only up to a plateau near the CMC. These experimental trends are well-described by a convective-diffusion model for surfactant transport to or from the liquid/liquid interface coupled with Langmuir-type adsorption, using a constitutive relation between the interfacial tension gradient and interfacial velocity based on the interfacial tangential stress jump.

Enabling Marangoni flow at air-liquid interfaces through deposition of aerosolized lipid dispersions.

It has long been known that deposited drops of surfactant solution induce Marangoni flows at air-liquid interfaces. These surfactant drops create a surface tension gradient, which causes an outward flow at the fluid interface. We show that aqueous phospholipid dispersions may be used for this same purpose. In aqueous dispersions, phospholipids aggregate into vesicles that are not surface-active; therefore, drops of these dispersions do not initiate Marangoni flow. However, aerosolization of these dispersions disrupts the vesicles, allowing access to the surface-active monomers within. These lipid monomers do have the ability to induce Marangoni flow. We hypothesize that monomers released from broken vesicles adsorb on the surfaces of individual aerosol droplets and then create localized surface tension reduction upon droplet deposition. Deposition of lipid monomers via aerosolization produces surface tensions as low as 1mN/m on water. In addition, aerosolized lipid deposition also drives Marangoni flow on entangled polymer solution subphases with low initial surface tensions (∼34mN/m). The fact that aerosolization of phospholipids naturally found within pulmonary surfactant can drive Marangoni flows on low surface tension liquids suggests that aerosolized lipids may be used to promote uniform pulmonary drug delivery without the need for exogenous spreading agents.

Alternative bioseparation operations: life beyond packed-bed chromatography.

Chromatography is undoubtedly the workhorse of downstream processes, affording high resolution for bioseparations. At the same time, it has the notoriety of being the single largest cost center in downstream processing and of being a low-throughput operation. Consequently, 'chromatography alternatives' are an attractive proposition, even if only a reduction in the extent of use of packed beds can be realized. This paper reviews the current state of unit operations posing as chromatography alternatives--including membrane filtration, aqueous two-phase extraction, three-phase partitioning, precipitation, crystallization, monoliths and membrane chromatography--and their potential to do the unthinkable.

Surfactant Driven Post-Deposition Spreading of Aerosols on Complex Aqueous Subphases. 1: High Deposition Flux Representative of Aerosol Delivery to Large Airways.

BACKGROUND: Aerosol drug delivery is a viable option for treating diseased airways, but airway obstructions associated with diseases such as cystic fibrosis cause non-uniform drug distribution and limit efficacy. Marangoni stresses produced by surfactant addition to aerosol formulations may enhance delivery uniformity by post-deposition spreading of medications over the airway surface, improving access to poorly ventilated regions. We examine the roles of different variables affecting the maximum post-deposition spreading of a dye (drug mimic). METHODS: Entangled aqueous solutions of either poly(acrylamide) (PA) or porcine gastric mucin (PGM) serve as airway surface liquid (ASL) mimicking subphases for in vitro models of aerosol deposition. Measured aerosol deposition fluxes indicate that the experimental delivery conditions are representative of aerosol delivery to the conducting airways. Post-deposition spreading beyond the locale of direct aerosol deposition is tracked by fluorescence microscopy. Aqueous aerosols formulated with either nonionic surfactant (tyloxapol) or fluorosurfactant (FS-3100) are compared with surfactant-free control aerosols. RESULTS: Significant enhancement of post-deposition spreading is observed with surfactant solutions relative to surfactant-free control solutions, provided the surfactant solution surface tension is less than that of the subphase. Amongst the variables considered--surfactant concentration, aerosol flow-rate, total deposited volume, time of delivery, and total deposited surfactant mass--surfactant mass is the primary predictor of maximum spread distance. This dependence is also observed for solutions deposited as a single, microliter-scale drop with a volume comparable to the total volume of deposited aerosol. CONCLUSIONS: Marangoni stress-assisted spreading after surfactant-laden aerosol deposition at high fluxes on a complex fluid subphase is capable of driving aerosol contents over significantly greater distances compared to surfactant-free controls. Total delivered surfactant mass is the primary determinant of the extent of spreading, suggesting a great potential to extend the reach of aerosolized medication in partially obstructed airways via a purely physical mechanism.