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The dilational modulus (E) of polymer films has been commonly measured using the oscillating ring/bubble/drop methods with an external force, and often without specifying the state of the adsorbed film. This study explores an approach where E was determined from the relaxations of surface tension (ST) and surface area (SA) of natural perturbations, in which ST and SA were monitored using a pendant bubble tensiometer. The E of the adsorbed film of PAA (polyacrylic acid) was evaluated for aqueous solutions at CPAA = 5 × 10−4 g/cm3, [MW = 5, 25, and 250 (kDa)]. The E (=dγ/dlnA) was estimated from the surface dilational rate (dlnA/dt) and the rate of ST change (dγ/dt) of the bubble surface from the natural perturbation caused by minute variations in ambient temperature. The data revealed that (i) a considerable time is required to reach the equilibrium-ST (γeq) and to attain the saturated dilational modulus (Esat) of the adsorbed PAA film, (ii) both γeq and Esat of PAA solutions increase with MW of PAA, (iii) a lower MW solution requires a longer time to reach its γeq and Esat, and (iv) this approach is workable for evaluating the E of adsorbed polymer films. [ABSTRACT FROM AUTHOR]

Two open issues on the measurement of the dilational modulus (E) for an adsorbed protein film during the adsorption process have been unacknowledged: how E varies during the adsorption and the length of time needed to attain a stable E value. A new approach for detecting the E variation from a clean air–water interface to saturated film and estimating the time needed to reach a saturated state was proposed. A pendant bubble tensiometer was utilized for measuring the relaxations of surface tension (ST) and surface area (SA), and the E was evaluated from the relaxation data of minute distinct perturbances. The data showed a clear variation in E during the BSA adsorption: E sharply decreased to a minimum at the early stage of BSA adsorption; then, it rose from this minimum and oscillated for a while before reaching an E corresponding to a saturated BSA film after a significant duration. The adsorbed BSA film took ~35 h to reach its saturated state, which was much longer than the reported lifetime of the adsorbed film in the literature. A rapid surface perturbation (forced bubble expansion/compression) could change the E, causing a significant drop in E followed by a slow increase to the original stable value. [ABSTRACT FROM AUTHOR]

Two open issues on the measurement of the dilational modulus (E) for an adsorbed protein film during the adsorption process have been unacknowledged: how E varies during the adsorption and the length of time needed to attain a stable E value. A new approach for detecting the E variation from a clean air–water interface to saturated film and estimating the time needed to reach a saturated state was proposed. A pendant bubble tensiometer was utilized for measuring the relaxations of surface tension (ST) and surface area (SA), and the E was evaluated from the relaxation data of minute distinct perturbances. The data showed a clear variation in E during the BSA adsorption: E sharply decreased to a minimum at the early stage of BSA adsorption; then, it rose from this minimum and oscillated for a while before reaching an E corresponding to a saturated BSA film after a significant duration. The adsorbed BSA film took ~35 h to reach its saturated state, which was much longer than the reported lifetime of the adsorbed film in the literature. A rapid surface perturbation (forced bubble expansion/compression) could change the E, causing a significant drop in E followed by a slow increase to the original stable value.

• Effect of surface area (SA) fluctuation on the dilational modulus (E) was studied. • A SA fluctuation (ΔA/A) larger than ∼3 % had a notable influence on e. • Distinct peak and minimum in e were detected when ΔA/ A > ∼3 %. • A smaller SA fluctuation (ΔA/ A < 3 %) caused only slight variation in E. • E rose to a distinct high after rapid compression, then decreased to a minimum. Understanding how surface area (SA) fluctuation affects the dilational modulus (E) of globular protein films is crucial for evaluating their mechanical properties and practical utility. However, limited reports are available in the literature and the measurement error is unclear. The impact of SA fluctuation and large, forced perturbation on the E of bovine serum albumin (BSA) and human serum albumin (HSA) film was investigated. The E was evaluated by analyzing surface tension (ST) and SA relaxation data obtained using a pendant bubble tensiometer. The data revealed that a large fluctuation in SA exerted a notable influence on the E : a distinct peak and minima in E being detected at the onset and end, respectively (at magnitude ΔA/ A > ∼3 % and at oscillation frequency ƒ = 0.3 – 1.1 mHz for adsorbed BSA/HSA film). In contrast, a comparatively smaller SA fluctuation caused only a slight variation in E. Furthermore, a large, forced perturbation led to a rise in E , exhibiting a distinctly high value after a rapid bubble compression, followed by a subsequent decrease to a distinctly low E. [Display omitted] [ABSTRACT FROM AUTHOR]

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N -myristoyl phenylalanine- N -polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

• New approach for evaluating the dilational modulus of protein films was proposed. • Relaxations of surface area and surface tension for a BSA aq solution were monitored. • Numerous minute interfacial perturbances were detected in the SA and ST relaxations. • Best-fits of relaxation data of these perturbances yielded the dγ/dt & dlnA/dt. • The modulus was evaluated from the slope of best-fit in dγ/dt vs dlnA/dt plot. A new approach for evaluating the dilational modulus of an adsorbed protein film was proposed in this study; wherein, the air-water interface of Bovine serum albumin (BSA) aqueous solution was not subjected to a forced perturbation. The approach involved: (i) monitoring the relaxations of surface tension (ST), surface area (SA), and ambient/solution temperature of aqueous bovine serum albumin (BSA) solution using a pendant bubble tensiometer and (ii) detecting the numerous minute and low-frequency interfacial perturbances in the SA and ST relaxations (resultant from the minute variations in ambient temperature) at the latter stages of BSA adsorption process. For these perturbances, their surface dilational rate (dlnA/dt) and rate of ST change (dγ/dt) were determined, and then the dilational modulus (E = dγ/dlnA) was obtained from the slope of the linear best-fit in the plot of dγ/dt vs dlnA/dt. Using this approach, the dilational modulus of the adsorbed BSA film at a concentration of 0.052 and 15 (10−10 mol/cm3) was found to be 43 and 30 mN/m, respectively. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dynamic surface tension (ST) of aqueous BSA solutions were measured for 2–5 days. • The ST remained nearly constant for several tens of hours during the latter stage. • Irrespective of BSA concentration, dynamic ST relaxed to a same value. • Forced and rapid bubble perturbations further confirmed these ST values. • From 0.05 to 15 (10-10 mol/cm3), the equilibrium ST kept constant at 51.5 ± 0.3 mN/m. The dynamic and equilibrium surface tension (ST) data of proteins such as bovine serum albumin (BSA) are crucial in numerous theoretical and practical applications. However, reliable equilibrium ST of BSA is absent in the literature. This study illustrates a more precise and accurate approach for measuring equilibrium ST of aqueous BSA solutions. The ST relaxation of aqueous BSA solution [C = 0.052–15 (10-10 mol/cm3)] was monitored using a pendant bubble tensiometer for 2–5 days, depending on the BSA concentration. The dynamic ST indicated that irrespective of the BSA concentration, the ST remained nearly constant at the latter stage for several tens of hours. These ST values were further confirmed by a perturbation process (a forced, rapid expansion/compression of the bubble surface). The data indicated that the equilibrium ST of BSA (aq) solutions remained essentially unchanged over a wide range of BSA concentration [C = 0.052 to 15 (10-10 mol/cm3)] at 51.5 ± 0.3 mN/m. [ABSTRACT FROM AUTHOR]

The pharmaceutical industry uses surface-active agents (excipients) in protein drug formulations to prevent the aggregation, denaturation, and unwanted immunological response of therapeutic drugs in solution as well as at the air/water interface. However, the mechanism of adsorption, desorption, and aggregation of proteins at the interface in the presence of excipients remains poorly understood. The objective of this work is to explore the molecular-scale competitive adsorption process between surfactant-based excipients and two monoclonal antibody (mAb) proteins, mAb-1 and mAb-2. We use pendant bubble tensiometry to measure the ensemble average adsorption dynamics of mAbs with and without the excipient. The surface tension measurements allow us to quantify the rate at which the molecules "race" to the interface in single-component and mixed systems. These results define the phase space, where coadsorption of both mAbs and excipients occurs onto the air/water interface. In parallel, we use X-ray reflectivity (XR) measurements to understand the molecular-scale dynamics of competitive adsorption, revealing the surface-adsorbed amounts of the antibody and excipient. XR has revealed that at a sufficiently high surface concentration of the excipient, mAb adsorption to the surface and subsurface domains was inhibited. In addition, despite the fact that both mAbs adsorb via a similar mechanistic pathway and with similar dynamics, a key finding is that the competition for the interface directly correlates with the surface activity of the two mAbs, resulting in a fivefold difference in the concentration of the excipient needed to displace the antibody.