Monoclonal Antibody Interfaces: Dilatation Mechanics and Bubble

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Monoclonal antibody interfaces: dilatation mechanics and bubble coalescence Aadithya Kannan, Ian C. Shieh, Danielle Lurisa Leiske, and Gerald G. Fuller Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03790 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Monoclonal antibody interfaces: dilatation mechanics and bubble coalescence Aadithya Kannan1, Ian C. Shieh2, Danielle L. Leiske3, and Gerald G. Fuller1* 1 Department of Chemical Engineering, Stanford University; 2 Late Stage Pharmaceutical Development, Genentech; 3 Early Stage Pharmaceutical development, Genentech

Abstract Monoclonal antibodies (mAbs) are proteins that uniquely identify targets within the body, making them well-suited for therapeutic applications. However, these amphiphilic molecules readily adsorb onto air-solution interfaces where they tend to aggregate. We investigated two mAbs with different propensities to aggregate at air-solution interfaces. The understanding of the interfacial rheological behavior of the two mAbs is crucial in determining their aggregation tendency. In this work, we performed interfacial stress relaxation studies under compressive step strain using a custom-built dilatational rheometer. The dilatational relaxation modulus was determined for these viscoelastic interfaces. The initial value and the equilibrated value of relaxation modulus were larger in magnitude for the mAb with a higher tendency to aggregate in response to interfacial stress. We also performed single-bubble coalescence experiments using a custom-built dynamic fluid-film interferometer (DFI). The bubble coalescence times also correlated to the mAbs aggregation propensity and interfacial viscoelasticity. To study the influence of surfactants in mAb formulations, polyethylene glycol (PEG) was chosen as a model

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surfactant. In the mixed mAb/PEG system, we observed that the higher aggregating mAb coadsorbed with PEG and formed domains at the interface. In contrast, for the other mAb, PEG entirely covered the interface at the concentrations studied. We studied the mobility of the interfaces, which was manifested by the presence or the lack of Marangoni stresses. These dynamics were strongly correlated with the interfacial viscoelasticity of the mAb's. The influence of competitive destabilization in affecting the bubble coalescence times for the mixed mAb/PEG systems was also studied.

Introduction Monoclonal antibodies (mAbs) are biologic therapeutics that are used in the treatment of a variety of diseases including cancer, respiratory conditions, and autoimmune disorders1-3. Aggregation of these mAb therapeutics is a serious processing challenge and may be induced by factors such as thermal stress, chemical modification, solvent effects, and freeze-thaw cycles4,5. A major cause of aggregation is related to the fundamental nature of these antibodies because they are amphiphilic and adsorb readily onto hydrophobic interfaces6,7. Antibody therapeutics encounter numerous hydrophobic interfaces during manufacture, storage, transportation, and administration8,9 including primary container walls, silicone oil droplets used for syringe plunger lubrication10,11, and air-solution interfaces in the form of entrained bubbles and headspace in vials or IV bags12,13. This adsorption, particularly at the air-solution interface, promotes aggregation under interfacial stresses8,14-15 and this aggregation can subsequently affect the drug quality16-17 and safety18-20 as well as possibly inducing anti-therapeutic antibodies in the body21-22 which reduce the efficacy of the drug.


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The intrinsic character of the protein plays an important role in the interfacial behavior of the molecule. For example, flexible proteins can unfold faster than globular proteins, affecting the rates of lowering of surface tension and the interfacial viscoelasticity23,24. The interfacial rheological properties of proteins have been well documented25-27. Similarly, colloidal properties, such as the charge distribution, are known to affect protein aggregation28. To study the effects of agitation on aggregation, traditional methods involve the shaking of vials29-30. In the past, our group pinpointed the major cause of aggregation to be interfacial dilatation (area-change) and not shear flow31. In order to perform experiments on a simple well-characterized system, single bubbles formed at the end of a capillary were chosen. This system also offered the advantage of allowing purely dilatational deformations unlike a Langmuir trough that has an equal-area extensional strain superimposed onto dilatation31. Interfacial stresses were determined by measuring the pressure inside a spherical bubble (diameter smaller than the capillary length). This approach was first used by Monteux et al.32 to study the mechanics of particle laden interfaces. Alvarez et al.33 developed a microtensiometer to measure surface stress across a spherical cap. In the primary packaging used for long-term storage of therapeutic mAbs12,13, air entrainment is inevitable. These air bubbles can rise to the top of a solution and coalesce with the flat airsolution interface created by the container headspace. This coalescence event causes a rapid distortion of the air-solution interface and may also promote shedding of surface-induced aggregates. There have been many investigations on bubble coalescence in the context of understanding foam stability 34-37 including the role of interfacial rheology38. There is significant literature on single bubble drainage39-44, specifically the drainage of a thin film using the Scheludko Cell geometry40,43,44. The influence of viscoelasticity imparted by proteins on thin


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film drainage has also been reported45-47. We recently developed the capability to study the thin film drainage dynamics as a bubble approaches a flat air-solution interface and the subsequent coalescence48. The dynamic fluid-film interferometer (DFI) can be used to study the drainage of liquid films during the process of bubble coalescence. Our work focuses on understanding how the adsorbed mAb layer at the interface affects the rheological behavior of the proteins upon interface compression as well as during thin film drainage followed by coalescence. We then relate these properties to the propensity of the antibody to aggregate. In this study, we used two mAbs, identified as mAb 1 and mAb 2, that share the same IgG1 framework and 93% sequence identity. MAb 1 has a higher tendency to aggregate on agitation compared to mAb 249,8. Past studies on the interfacial behavior of these mAbs revealed that the surface excesses (surface concentrations) of both mAbs saturated in less than 90 s, which was determined through fluorescent- labeling of the antibodies. The surface excess saturated to values of 420 ng/cm2 and 220 ng/cm2 for mAb 1 and mAb 2, respectively. Hence, the surface concentration of mAb 1 was approximately twice that of mAb 2. The surface pressure, however, continued to increase at film ages beyond 30 min showing the predominance of intermolecular rearrangement over surface concentration on influencing the surface pressure. More interestingly, the intramolecular unfolding of these mAbs was studied using Nile red, which fluoresces in nonpolar, hydrophobic environments50. At the interface, mAb 1 rapidly unfolded and exposed hydrophobic regions of the protein likely leading to strong intermolecular interactions. In contrast, mAb 2 required more residence time on the air-solution interface before unfolding.49 Surfactants are typically used in pharmaceutical formulations as they competitively adsorb onto the interface and prevent the adsorption of proteins that leads to aggregation.51,10,12,13,30. In


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the past, there have been studies on the interfacial rheological behavior of these proteinsurfactant mixtures52-54. In this work, we also studied mixed mAb-surfactant systems using polyethylene glycol as the surfactant to elucidate the influence of competitive adsorption by surfactants on interfacial compression and bubble coalescence.

Experimental Section Preparation of mAb and PEG solutions Two antibodies (referred to as mAb 1 and mAb 2) were produced in Chinese Hamster ovary cells and purified at Genentech. MAb 1 and mAb 2 share the same IgG1 framework with κ light chains, and both have a molecular weight of approximately 145 kDa. The antibodies differ only in their complementarity determining regions and share 93% sequence identity. Mab1 and mAb2 have theoretical isoelectric points of 9.0 and 9.1, respectively. The mAbs were obtained from ultrafiltration/diafiltration pools and were free of surfactants. Solutions at 0.2 mg/mL mAb were prepared using a low ionic strength buffer at pH 5.5 provided by Genentech. Polyethylene glycol with a molecular weight of 10 kDa (Sigma-Aldrich 92897-1KG-F) was used as the surfactant added to the mAb solution where indicated. Dilatational rheometry A custom-built dilatational rheometer was used to study the rheological properties of airsolution interfaces. Details of the setup along with a schematic is provided in a previous work31. A glass chamber (3 cm x 3 cm x 2 cm) was filled with 5 mL of mAb solution at 0.2 mg/mL, and a downward facing silica glass capillary (Polymicro, OD: 665 µm, ID: 536 µm, TSP530660) was immersed in it. This was connected to a syringe pump (Harvard Apparatus Pump 11 Elite HA1100W; 1802 25 µL syringe, barrel diameter = 0.732 mm: Hamilton, USA) and a differential pressure transducer (Omegadyne PX409-10WGUSBH). The image of the bubble that is created


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was captured from the side using a camera (Basler ace (CCD) camera, acA640-90gm, 659 x 494, 90 fps, Monochrome). A MATLAB script controlled the syringe pump and periodically collected images from the side camera and extracted information about the bubble surface area and volume using a spherical cap formula. During an experiment, a bubble was blown to a volume of 1.5 µL taking into considerations the critical volume required to create a stable bubble55. It was aged for 30 s – 1800 s and then a step compression was applied (Figure 1a). Since we are interested in aggregation, and a compression of the interface is the mode of area change that has a higher possibility of inducing aggregation15, we performed bubble contraction experiments on the airsolution interface. During the relaxation of the bubble, the pressure was recorded and simultaneously the bubble shape information was collected. The transducer measured a pressure that is the sum of pressure drop across the interface and the hydrostatic pressure. Based on the depth of immersion, the hydrostatic head was subtracted and ∆ = − , where Ptot is the total gauge pressure and Ph is the hydrostatic pressure. Assuming the bubble maintains a spherical shape, a normal stress balance gives ∆ = 2 / where is the interfacial stress and R is the bubble radius. An apparent excess stress is defined as ∆ = − , the difference between the stress prior to interfacial compression, and the measured interfacial stress during relaxation. The strain is defined as = ∆/ where ∆ = − and is the area prior to compression and is the area at any later time. A strain of ~0.15 (within the linear viscoelastic regime) was applied to the interface. The dilatational relaxation modulus is defined, = ∆ /. This quantity incorporates the deviatoric stresses along with the isotropic contributions of surface tension. The relaxation after the step compression is applied has a single exponential behavior (Figure 1b) where the peak of the decay is defined as the initial dilatational modulus (E0) and the long


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time, equilibrated value is defined as the static dilatational modulus (E∞). Proteins irreversibly adsorb to interfaces25,31, so we have neglected bulk diffusion and have assumed the number of molecules adsorbed does not change on compression in our definition of the static dilatational modulus. The relaxation behavior is characteristic of a viscoelastic solid interface that fits with a Standard Linear Solid (Kelvin model). Physically, the initial modulus represents the stress-strain response of the viscoelastic network and is proportional to the number of aggregation linkages in the adsorbed film. Once deformed, the strained network will relax the imposed stress by rearrangements that ultimately diminish both the number of linkages and the interlinkage deformations. These relaxation processes lead to a static relaxation modulus. This is represented in Figure 1, a and b. The difference between the two, ∆ = − , is defined as the dissipation dilatational modulus. Similar definitions have been used in the past56. The exponential fit performed using MATLAB’s non-linear least squares fitting algorithm yielded a relaxation time, τr, corresponding to the decay.

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Figure 1. a) Experimental procedure in a step-compression experiment with a visual representation of possible behavior of proteins at the interface during aging, after step compression and during relaxation. b) A theoretical plot of dilatational relaxation modulus during a step-compression experiment with initial, static, and dissipation moduli indicated. c) Dynamic fluid film interferometer with the d) final position of the bubble depicted during drainage and eventual coalescence. e) An example top view image showing the interference pattern and the thicker region at the apex which formed a dimple. f) Internal bubble pressure trace during a typical coalescence experiment with the different stages identified.

Thin film drainage and bubble coalescence studies


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The dynamic fluid-film interferometer (DFI) (Figure 1c) consisted of a Delrin chamber containing the protein solution. A 16-gauge blunt-tipped needle (1.194 mm I.D; 1.651 mm O.D.) was inserted through a silicone elastomeric membrane (Marian Chicago, Inc. HT6220) at the bottom. The needle was connected to a pressure transducer and a gas tight syringe actuated using a syringe pump. The bubble was imaged through a side camera (ThorLabs DCU223C) and a top camera (Imaging Development Systems UI-3060CPC). A motor (Newport CMA 25PP and SMC100PP) positioned the bubble relative to the flat air-solution interface at the top of the solution. A light source above the interface (CCS Inc. LAV-80SW2) induced reflection interference and was filtered using a dichroic filter (Edmund Optics #87245) with pass bands of 457 nm, 530 nm, and 628 nm. In a coalescence experiment, the chamber with the protein solution was lowered towards a bubble of volume 2.5-3.0 µL. This caused the bubble interface to approach the flat air-solution interface at a velocity of 0.15 mm/s. The flat interface continued to move lower until the apex of an undeformed bubble protruded a distance of half of the bubble radius above the flat interface as shown in Figure 1d and e. A typical pressure trace is shown in Figure 1f. The initial flat region of the pressure trace occurred when the bubble was stationary. As the bubble approached the interface, the pressure decreased in response to the decreasing hydrostatic head. The subsequent increase in pressure was a manifestation of the deformation of the bubble as it was pressed against the interface48. This deformation is depicted in Figure 1d where a cartoon image of an actual bubble is shown in relation to an undeformed bubble. The following plateau region was where drainage of the film occurred. Coalescence of the bubble was manifested by a sudden drop in the pressure and this determined the coalescence time of the experiment (Figure 1f). Confocal imaging


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MAbs fluorescently labelled with Alexa Fluor-488 were obtained from Genentech. Details of the labelling procedure is described elsewhere8,49. Borosilicate glass coverslips (3 in. × 1 in. × 170 µm) were cleaned with a 2% solution of Hellmanex III (Hellma Analytics 9-307-011-4-507), rinsed with DI water, and then placed in a UV-ozone cleaner for 20 min. This treatment rendered the surfaces of the coverslips more hydrophilic enabling spontaneous spreading of mAb solutions. Subsequently, an 8-well slide cover (Ibidi 80828) was positioned on the coverslip. The lid of the well was fitted with a moist filter paper to limit evaporation during the imaging. A volume of 15 µL of mAb solution with surfactants at different concentrations was spread on the coverslip in each well. A Leica TCS SP5 inverted laser scanning confocal microscope was used to measure fluorescence at the air−liquid interface during film aging through a 20×, 0.7 N.A. water immersion objective. A z-stack of 100 images was acquired at 0.25 µm steps through the interface using a pinhole of 0.5 Airy units for optimal axial resolution. Z-stacks were recorded as a function of time for up to 30 min after initial spreading of the film.

Results and Discussion Step-compression studies Step-compression experiments highlighted the different viscoelastic behavior of interfacial films of mAb 1 and mAb 2, and demonstrated that the antibody films with higher moduli values corresponded to the mAb with the higher aggregation propensity. Figure 2a shows the behavior of the initial dilatational modulus, E0, of mAb 1 and mAb 2 films at the air-solution interface as a function of the adsorbed film age (age time) up to 30 minutes. MAb 1, the antibody with greater aggregation propensity, exhibited a higher magnitude of initial dilatation modulus at all film ages compared to mAb 2. The initial modulus represents the accumulation of elastic energy during compression. The higher initial modulus for mAb 1 at all film ages suggests that this antibody


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forms a more cohesive elastic network, with more aggregation linkages, at the air-solution interface, which is likely a result of more extensive unfolding of mAb 1 compared to mAb 2. The higher surface excess reported for mAb 149 (420 ng/cm2 vs 220 ng/cm2 for mAb 2) would also contribute to the formation of a network with more aggregation linkages. For both the mAbs, the modulus increased as a function of the aging time and, after an initial fast rate of increase, continued to grow at more modest rates. This behavior was consistent with previous reports demonstrating that antibody films quickly saturate in surface excess (concentration) but, through processes of antibody unfolding and rearrangement, interfacial properties continue to evolve.8,49

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Figure 2. a) Initial Dilatational Modulus, E0, increases with age time and is higher for mAb 1 b) Static Dilatational Modulus, E∞, is higher for mAb 1 c) Dissipation Dilatational Modulus, ∆E, is


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similar for both the mAbs d) Relaxation Time, τr, is higher for mAb 2 but is increasing at low age times, whereas for mAb 1 it is constant.

A similar trend was seen for the static dilatational modulus, E∞ (Figure 2b), highlighting that both the initial response and eventual relaxed state after a step compression correlated with the tendency of the proteins to aggregate. The static modulus represents the degree of irreversibility in an antibody film after a step compression. This reinforces the hypothesis that the more cohesive elastic network will relax to a state that is more distinct from the pre-compression state than a network that is not as cohesive. During relaxation, the accumulated elastic energy may be dissipated by the formation of loops and tails of the segments of the antibodies protruding into the water phase56. In contrast to the initial and static moduli, the dissipation dilatational modulus, ∆E was similar for both mAbs (Figure 2c). As the antibody film at the air-solution interface aged, the initial modulus increased faster than the increase in static modulus, as shown by the increase in dissipation modulus with film age. Since the antibody films formed a more wellconnected network with interface age, the differences in surface stress of the strained network were more prominent than the equilibrium state to which it eventually relaxed. Figure 2d shows the behavior of relaxation times of the mAb solutions as a function of aging time after fitting to a Kelvin model for a viscoelastic solid with a single exponential decay31. MAb 2 exhibited a higher value of the relaxation time than mAb 1. Interestingly, the relaxation times of mAb 1 did not show an aging time dependence. However, for mAb2, the relaxation time increased initially with aging time and then reached a constant value. Note that in this plot, the 120 s, 150 s, and 180 s aged experiments were grouped together under the 150 s case to show this trend. MAb 1 had a lower relaxation time than mAb 2, which seemed counter intuitive as


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compared to the moduli behavior. This was possibly because mAb 1 had a time scale of intrinsic rearrangement faster than mAb 2 as also reflected in the rate of increase of surface pressures8 and the rate of unfolding of the mAbs49. MAb 1 has a higher surface excess than mAb 2 and on compression, mAb1 is packed more tightly and the relaxation could be more constrained to a shorter distance and thus occur faster. The difference in the relaxation times observed may also be due to a different relaxation mechanism for the two mAbs. MAb 1, which has a higher surface excess and possibly more aggregation linkages, may form more loops and tails upon compression that protrude into the subphase. During relaxation, these loops may protrude further away from the interface to further relax the stress in the film instead of intermolecular rearrangement within the network. This would also explain the time-independent relaxation seen for mAb 1. The time scale of saturation of the relaxation time for mAb 2 correlated with the time scale of unfolding of the same mAb as reported by Leiske et al.49. Liquid film drainage and coalescence Studies on thin liquid film drainage and coalescence as a bubble approached an interface revealed qualitative differences in behavior in the presence of viscoelastic interfaces. Using the dynamic fluid-film interferometer, bubble drainage and coalescence studies were performed for antibody films aged for 30 s, 300 s, and 900 s. The 30 s experiments represented the minimum time required to initialize the experiments. The 300 s case represented the situation when mAb 1 was considerably unfolded at the interface but mAb 2 was not, as reported by Leiske et al.49. Finally, the 900 s case was when mAb 2 was also significantly unfolded and, more importantly, both the mAbs were rearranging and forming a network as the surface pressure continued to increase8,49. As the air bubble in the liquid approaches the top air-liquid interface of the chamber, a thicker fluid region, a dimple41(Figure 1e), is trapped at the apex because of the capillary


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pressure deforming the interface. This dimple is unstable and washes out to the sides after the bubble stops moving. However, surface viscoelasticity can stabilize the dimple dynamics to the point that washout does not occur. This stabilization occurred in the presence of both mAb 1 and mAb 2 (Supplementary video 1). The presence of particulate matter can be found in the interference patterns that identify the production of mAb aggregates (Figure 3a). At longer aging times, the fluid film drained for a longer period before coalescence to the point where a black fluid film was observed signifying a fluid film thickness of < 50 nm (Figure 3b). The presence of a black film in a surfactant solution is usually attributed to the effect of the electrostatic component of the disjoining pressure of the adsorbed film57. In contrast, the interactions can be hydrophobically driven for proteins at such small thicknesses, and the presence of monolayers or multilayers of proteins with very little or no solvent has been observed previously58.

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Figure 3. Screenshots during different stages of thin fluid film drainage. a) Interference pattern with a stationary dimple at the center seen in mAb 1 solution at 30 s interface aging time when the bubble stopped moving. b) The black film seen with mAb 1 at 900 s interface aging time after more than 30 s of thin fluid film drainage without coalescence.


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The coalescence time of a bubble at the air-solution interface corresponded to the viscoelasticity and stability of that interfacial mAb film and provided additional insight into the aggregation propensity of the mAbs in that film. The coalescence times for mAbs 1 and 2 at different film ages are represented in Figure 4. The coalescence time for both the mAbs increased with aging time, again suggesting the formation of interconnected protein networks at the air-solution interface. This increase in coalescence time with film age was consistent with the increase in initial modulus during step-compression experiments (Figure 2a). For higher aging times, there was significant variation in the coalescence time values as reflected in the standard deviations (Figure 4). This was likely due to the particles formed and entrained between the two antibody films which generally increased the viscoelasticity of the interfaces, and hence increased the coalescence time. These aggregates are particles of proteins with spatially localized hydrophobic and hydrophilic regions. Based on the local contact of the air-solution interface with the particle aggregates and the wetting behavior, they can also bridge the two interfaces and thus act as antifoaming agents. We can observe that the coalescence time of mAb 1 was higher than mAb 2 at the three aging times studied. Also, in the presence of the black film consistently seen for the mAb 1 solutions at higher age times, when the needle was withdrawn, adhesion of the bubble to the flat interface was observed. Details about this adhesion behavior will be reported in a future publication. This adhesion suggests the possibility of having a very stable cohesive network across the two air-solution interfaces and is likely related to the higher tendency of mAb 1 to aggregate.


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Figure 4. Coalescence Time of mAb 1 and mAb 2 solutions at interfacial film ages of 30, 300, and 900 s. Coalescence time increased with film age and a higher coalescence time was measured for mAb 1 at all film ages.

Competitive adsorption of mAbs with surfactants Therapeutic mAb formulations typically include a surfactant to stabilize the protein against interfacial stress through competitive adsorption. Polyethylene glycol (PEG), which exhibits surface activity59-62, was chosen as a weak surfactant to better understand the competitive adsorption between surfactants and mAbs. Confocal imaging of the air-mAb/PEG solution interface (Figure 5) shows the co-adsorption of the fluorescently labelled mAb (green) and PEG (black) at the interface. The percentage of PEG included with the mAb solution refers to the weight of PEG in a unit volume of solution (e.g., 0.01% PEG is 0.1 mg/mL of PEG in solution). Figure 5 shows that mAb 1 co-adsorbed with PEG to form discrete domains with more surfactant coverage (dark regions) at higher surfactant concentrations. In contrast, mAb 2 was unable to adsorb onto the interface in the presence of PEG, demonstrated by the absence of any fluorescence signal.


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Figure 5. Confocal images of mAb+PEG at two concentrations and film ages. Antibody was fluorescently labelled and is represented by green intensity. MAb 1 competitively adsorbed with PEG with increasing mAb surface coverage with film age whereas with mAb 2 the interface was entirely covered with PEG.

The co-adsorption of mAb 1 with PEG observed using confocal microscopy also manifested both in step-compression experiments as well as in coalescence experiments.


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Figure 6 shows the behavior of the static dilatational modulus, a representation of the residual elasticity after rearrangement and relaxation, for mAbs 1 and 2 in the presence of PEG at 0.01 % at an age time of 900 s. MAb 1+ 0.01% PEG had a lower E∞ than pure mAb 1 at the same aging time (~ 20 mN/m versus ~ 50 mN/m), but was non-negligible in contrast to a pure PEG film. MAb 2+ 0.01 % PEG exhibited the same relaxation behavior as pure PEG, which indicated that the interface was not viscoelastic and was dominated by PEG rather than mAb 2.

Figure 6. Static dilatational modulus of mAbs with PEG at an interfacial film age of 900 s. The finite value of mAb 1+PEG indicates competitive adsorption of mAb with PEG, whereas mAb 2+PEG is indistinguishable from a pure PEG solution.

The addition of PEG to mAb solutions and the disruption of the viscoelasticity was reflected in the mobility of the interfaces sandwiching the liquid thin film during drainage and coalescence experiments. For a pure mAb solution, the dimple was stationary as mentioned earlier. For a pure PEG solution, as the bubble protruded into the flat air-solution interface, more area was created at the apex due to the interface deformation. This region had a depleted surfactant concentration and hence a higher surface tension. The resulting surface tension gradient pulled surfactant


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molecules from the periphery along with the solution causing an instability and plumes of liquid to flow as shown in Figure 7a and Supplementary video 2. Because of the influx of solution into the thin fluid film, the dimple became unstable and washed out to one of the sides (Figure 7b).

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Figure 7. Snapshots of the interference pattern of 300 s interfacial film age time experiments taken approximately 3 s after the bubble stopped moving. a) Inward flows of solution into the thin film as a result of Marangoni stresses for pure PEG (0.01%) leading to b) dimple washout to the sides. Representative dimple dynamics for c) mAb 1 + 0.001% PEG, d) mAb 1 + 0. 01% PEG, e) mAb 2 + 0.001% PEG, and f) mAb 2 + 0.01% PEG.


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For mAb + PEG systems, we observed a correspondence between the interfacial mobility and mAb-PEG surface coverage. Figure 7, c-f shows snapshots of the dimple dynamics for the mixed systems at a 300 s interfacial film age. MAb 1 + 0.001% PEG had an immobile dimple whereas mAb 1 + 0.01% PEG, mAb 2 + 0.001% PEG, and mAb 2 + 0.01% PEG all exhibited a mobile dimple that washed out to the sides. Figure 8 quantifies the dimple washout dynamics and shows the behavior of the average dimple velocity (time average of the instantaneous velocities of the dimple). From the confocal images for mAb 1 (Figure 5), we observed an increase in surface coverage of the antibody with aging time. Confocal images of 30 s age could not be obtained due to experimental limitations in setting up the imaging within 30 s of creating an interface. Nevertheless, the surface concentration of mAb continued to increase for longer times and therefore, by extrapolation, would have been lower at a 30 s age compared to 300 s. For mAb 1+ 0.001% PEG, dimple washout (non-zero average velocity) only occurred for the 30 s case. In the 300 s and 900 s cases, the dimple remained stationary analogous to a pure mAb case. For a higher surfactant concentration (0.01% PEG), both the 30 s and 300 s cases exhibited dimple washout as the interface maintained its fluidity for a longer time. For mAb 2, the interface was entirely covered with PEG, and the mAb surface concentration did not change with interfacial film age. This was seen as the dimple always washed out for both PEG concentrations at all three age times studied.


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Figure 8. Dimple washout velocity for mAb 1 and mAb 2 in the presence of PEG. A non-zero velocity indicated mobility of the dimple, which was not observed for a pure mAb interfacial film. For mAb 1 + PEG at higher aging times, the interfaces became sufficiently immobile and the dimple was trapped.

MAb solutions substantially stabilized the draining thin film (i.e., had longer coalescence times than pure water [approximately 1-4 s]) as the dimple was immobilized by interfacial viscoelasticity. In the case of a pure surfactant solution (i.e., PEG), the mobile surfactants at the interface induced Marangoni flows that opposed drainage and lead to longer coalescence times (Supplementary video 2). The mixed mAb +PEG system exhibited lower coalescence times than the pure mAb system due to competitive destabilization54. The interfacial mobility of the PEG that caused Marangoni flows was inhibited by mAb and, at the same time, the interfacial viscoelasticity of the mAb film was disrupted by the presence of the PEG. Evidently, on adding PEG to mAb solutions, the reduction in viscoelasticity was more important than the increase in Marangoni stresses in controlling the coalescence time. Figure 9 shows the coalescence time behavior of mAb + PEG systems, at PEG concentrations of 0.001 % and 0.01 %, compared to


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pure mAb and pure PEG. For a mAb 1 + PEG interface aged for 900 s, as the concentration of PEG increased, the coalescence time decreased. For mAb 2, at 900 s age, the coalescence times were similar to the pure PEG case (dashed lines). This coalescence time behavior also shows a correspondence with the fractional coverage of mAb vs PEG as evident in the confocal images (Figure 5 for 900 s age). While PEG effectively kept mAb 2 from adsorbing to the air-liquid interface, mAb 1 was able to co-adsorb with PEG especially at longer interfacial film ages and lower PEG concentrations. This behavior was also supported by the confocal images and dimple dynamics presented earlier.

Figure 9. Coalescence time of mAb 1 and mAb 2 in the presence of PEG at 0.001% and 0.01% PEG. Solid red lines show the coalescence time for a pure mAb solution at that film age and the dotted lines show the coalescence time for a pure PEG solution. A dependence on concentration on the coalescence time was seen for mAb 1 at higher aging times.

Conclusions


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The viscoelasticity of a monoclonal antibody solution interface depended on how long the interface has been aged and the nature of the mAb molecule itself. This was manifested in two behaviors: -1) response to a step strain compression and 2) film drainage and coalescence when a bubble is brought against a flat air-solution interface. The relaxation modulus of a mAb solution-air interface on step compression showed an exponential decay behavior. The peak in the relaxation modulus immediately following a step compression, referred to as the initial dilatational modulus, and its final relaxed value, the static dilatational modulus, were quantified and were seen to increase with aging time and to be higher for mAb 1 compared to mAb 2. The coalescence time of a bubble in a mAb solution that was pushed against a flat air-solution interface was higher for mAb 1. This behavior correlated well with the higher propensity of mAb 1 to aggregate at the air-solution interface. When a surfactant (i.e., PEG) was added to this system, the surfactant competitively adsorbed onto the air-solution interface reducing the ability of the protein to form a strong viscoelastic network. This lowered the relaxation moduli values and the coalescence times. It increased the mobility of the interface resulting in the washout of the dimple of fluid formed during coalescence experiments. Further experiments showed the effect of surface coverage of mAb and surfactant solution in the coalescence times of bubbles. In the pharmaceutical industry, surfactants are added at a sufficient concentration in formulations to essentially completely cover the interface with surfactants to prevent protein adsorption. This work demonstrated that even at lower surfactant concentrations, where there is significant co-adsorption of the mAb with the surfactant, the reduction in interfacial viscoelasticity may reduce agitation-induced aggregation of the mAb.


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In conclusion, the tendency of mAbs to aggregate due to interfacial stress and the change in aggregation behavior caused by addition of surfactants can be successfully correlated to wellcontrolled dilatational and coalescence experiments.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Supplementary Video 1: Thin film drainage observed from the top camera of the dynamic fluidfilm interferometer. A bubble in mAb 1 solution is aged for 900 s and pushed against the flat interface (.mp4) Supplementary Video 2: Thin film dynamics and dimple washout observed from the top camera of the dynamic fluid-film interferometer. A bubble in polyethylene glycol solution aged for 300 s and pushed against the flat interface (.mp4) AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS


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We thank Gigi Lin and John Frostad for helpful discussions and support in performing the experiments. We thank Genentech for financial support and supply of the materials used in this work. REFERENCES 1.

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Table of Contents Graphic


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Monoclonal Antibody Interfaces: Dilatation Mechanics and Bubble

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