Article pubs.acs.org/molecularpharmaceutics
Predicting the Agitation-Induced Aggregation of Monoclonal Antibodies Using Surface Tensiometry Ian C. Shieh and Ankit R. Patel* Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States S Supporting Information *
ABSTRACT: Adsorption of antibody therapeutics to air−liquid interfaces can enhance aggregation, particularly when the solution does not contain protective surfactant or when the surfactant is diluted as occurs during preparation of intravenous infusion bags. The ability to predict an antibody’s propensity for interfacially mediated aggregation is particularly useful during product development to ensure the quality, potency, and safety of the therapeutic. To develop a predictive tool, we investigated the surface pressure and surface excess of a panel of 16 antibodies as well as determined their aggregation propensity at the air−liquid interface in an agitation stress model. Our data demonstrated that the initial rate of surface pressure increase upon antibody adsorption to the air−liquid interface strongly predicted the extent of agitation-induced aggregation. Other factors, including the hydrophobicity, equilibrium surface pressure, and interfacial concentration of an antibody, were not adequate predictors of its susceptibility to aggregation. In addition to developing a predictive tool, we extended the interfacial characterization to better understand the mechanisms of antibody aggregation at an air−liquid interface during agitation stress. We believe that the kinetics of antibody rearrangement and conformational change after adsorbing to the interface, leading to the development of attractive antibody−antibody interactions, dictated the extent of aggregation. Overall, our results demonstrate how surface pressure measurements can be implemented as a rapid screening tool for the identification of antibodies with a high propensity to aggregate upon adsorption to an air−liquid interface while also furthering our understanding of interfacially mediated protein aggregation. KEYWORDS: air−liquid interface, aggregation, agitation stress, monoclonal antibodies, surface pressure, surface excess, pharmaceutical development
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INTRODUCTION
In this study, we focus on mAb aggregation enhanced by adsorption to the air−liquid interface created by the headspace in bags used for intravenous (IV) administration.14−16 Formation of aggregates in IV bags is particularly deleterious as there are typically limited options for removing them. While in-line filtration during IV administration can remove protein particles, it is not capable of removing soluble aggregates. During IV bag preparation, dilution of the drug product into the infusion bag dilutes the surfactant included in the original formulation, which reduces the effectiveness of the surfactant in competitively limiting the interfacial adsorption of mAb.17 Agitation of the air−liquid interface during transportation, such as when IV bags are transferred from the compounding pharmacy to the patient, can continuously regenerate the air− liquid interface, thereby increasing the quantity of mAb exposed to the interface and exacerbating aggregation. Previous reports have indicated that (1) the extent of agitation-induced aggregation is directly correlated with the amount of air−liquid interface generated,18 (2) compression of a mAb film at the air−liquid interface leads to the formation of particles,19 and
Monoclonal antibodies (mAbs) have rapidly emerged as a highly effective class of therapeutics over the past several decades.1,2 During development of therapeutic mAbs, the tendency of the proteins to aggregate is a concern because such aggregation can affect product quality, potency, safety, and immunogenicity.3−5 Although the extent of the risk that aggregates pose remains unclear,6−8 it is important during development to identify mAb candidates with a tendency to aggregate to implement appropriate risk mitigation strategies. Aggregation of therapeutic mAbs can be induced by numerous factors such as long-term storage at moderate or high concentrations, chemical degradation, interactions with excipients or leachables from containers, as well as thermal, oxidative, and interfacial stresses.9,10 Interfacial stresses are commonly encountered by mAb therapeutics during the manufacture, storage, and clinical use of the product and include stresses that occur at liquid−liquid, solid−liquid, and air−liquid interfaces. For example, adsorption of mAbs to droplets of silicone oil used to lubricate the plunger in prefilled syringes can enhance antibody aggregation.11,12 Interactions with solid interfaces can also enhance formation of aggregates during pumping, storage, and freeze−thaw cycles, which induce ice−water interfaces.13 © 2015 American Chemical Society
Received: Revised: Accepted: Published: 3184
January 28, 2015 May 29, 2015 July 21, 2015 July 21, 2015 DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
Article
Molecular Pharmaceutics
other interfacially adsorbed molecules and with molecules in the subphase all affect the surface pressure of a mAb film. In addition to equilibrium surface pressure, the kinetics of surface pressure changes were investigated to determine how interfacial dynamics affected aggregation during agitation. Finally, hydrophobic interactions are hypothesized to drive aggregation; thus, we examined if either a measured or a theoretical hydrophobicity for the mAbs predicted aggregation risk. The work presented here is the first example of the successful use of interfacial properties to predict the extent of agitation-induced aggregation in therapeutic mAbs. Beyond air−liquid interfaces, our approach could potentially be extended to mAb aggregation mediated by liquid−liquid and solid−liquid interfaces.
(3) eliminating the air−liquid interface by removing the headspace in the IV bag essentially prevents agitation-induced aggregation.15,17 Also, the percentage of total protein that aggregates has been shown to decrease with increasing bulk protein concentration,20 supporting the theory that aggregation during agitation is primarily interfacially mediated. Although the exposure of biotherapeutics to air−liquid interfaces has clearly been identified as a risk factor for aggregation, the mechanisms through which interfaces induce aggregation remain unclear. It is hypothesized that mAbs and other proteins adsorbing to the air−liquid interface undergo changes in conformation or orientation due to the hydrophobic environment presented by the air. These changes can lead to the exposure of hydrophobic portions of the proteins, which then enhance aggregation. However, variations in the physical properties of proteins and the formulation conditions lead to diverse behavior at the air−liquid interface. Early studies comparing the effects of shaking on a panel of proteins demonstrated the wide spectrum of interfacially mediated, agitation-induced aggregation behavior.21 Intrinsic protein properties can greatly affect their interfacial behavior; for example, the flexible protein β-casein was shown to increase surface pressure (i.e., reduce surface tension) immediately upon adsorption to the air−liquid interface, which suggested rapid unfolding, compared to the globular protein lysozyme that likely did not rapidly unfold and had a lag between adsorption and surface pressure increase.22 Colloidal properties of protein solutions can also influence interfacial aggregation. A variety of anions were shown to induce different effective charges on a panel of three mAbs, leading to variations in the extent of agitation-induced aggregation.23 Because of the many parameters that can affect interfacial protein aggregation, it is a difficult process to predict. During development of a therapeutic mAb, a tool for identifying candidate molecules at risk for interfacial aggregation would be extremely useful during molecule selection and assessment. Biopharmaceutical companies typically assess interfacial aggregation risk of a specific mAb using vial or IV bag agitation studies during formulation development.15 However, these studies can require large quantities of mAb (approximately 100−1000 mg per condition), which may not be readily available during early development. In this study, we aimed to develop a predictive tool for assessing interfacial aggregation risk by evaluating the interfacial properties of a panel of 16 therapeutic mAbs and the propensity of the mAbs to aggregate in an agitation stress model. In particular, we examined the equilibrium surface excess and surface pressure of the mAb films. The surface excess, which is the two-dimensional concentration of antibody at the air−liquid interface, was investigated to determine if aggregation depended strongly on the amount of mAb adsorbed to the interface. Surface pressure, Π, is the decrease in surface tension of a clean air−liquid interface: Π = γ0 − γ
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EXPERIMENTAL SECTION Antibody and Solution Preparation. An Elga PURELAB Ultra system provided ultrapure water with a resistivity of 18.2 MΩ cm for this work. Sixteen unique, full-length IgG antibodies were produced in Chinese hamster ovary cells and purified at Genentech. All mAb material was obtained from ultrafiltration/diafiltration pools prior to conditioning and was essentially free of surfactant. The number identifying each mAb corresponds to its rank order for interfacial aggregation propensity (i.e., mAb-1 showed the most aggregation). Prior to use, the mAbs were extensively dialyzed against a low ionic strength buffer at pH 5.5 to ensure consistent solution conditions across all antibodies. Concentration of the stock mAb solutions was measured by UV absorbance at 280 nm using known extinction coefficients ranging from 1.27−1.70 mg/mL/cm. Fluorescent Labeling of Antibodies. Alexa Fluor 488 (AF488; excitation/emission maxima at 494/515 nm) was covalently attached to the mAbs using a nonspecific lysine labeling procedure. Prior to labeling, mAb stock solutions were diluted to 10 mg/mL with 0.1 M NaHCO3 and then dialyzed for 2 h against 0.1 M NaHCO3. A total of 20 mg of the dialyzed mAb was then mixed with 100 μg of lyophilized AF488 2,3,5,6tetrafluorophenyl ester (Life Technologies; catalog number A37570) for a ratio of 0.8 mol AF488:mol mAb. The reaction mixture was incubated for 1 h at room temperature with gentle stirring and then extensively dialyzed against a low ionic strength buffer solution at pH 5.5 to remove excess dye. The fluorescent mAb material was characterized to ensure a low degree of labeling, proper removal of excess fluorophore, and limited aggregation. Concentration of the AF488-mAbs was measured using absorbance spectroscopy at 280 nm correcting for the absorbance of the AF488 at 280 nm. The correction was performed by measuring absorbance at 494 nm and using the manufacturer-supplied A280/A494 ratio of 0.11 for AF488. Size-exclusion chromatography (SEC) was used to assay the degree of labeling, free dye, and soluble aggregates for the AF488-mAbs. An Agilent 1100/1200 series HPLC equipped with a diode array detector and a Tosoh TSKgel G3000SWxl column (catalog number 08541) was used to perform sizing analysis. AF488-mAbs were injected at 1 mg/mL and eluted isocratically for 30 min at 0.5 mL/min using a mobile phase of 0.2 M K3PO4 and 0.25 M KCl (pH 6.2). Postcolumn absorbance was monitored at both 280 and 494 nm. The AF488-mAbs did not show an increase in soluble aggregates compared to the unlabeled mAb material. Degree of labeling of the AF488-mAbs was determined by comparing the integrated areas of the monomer peak detected at 494 nm versus at 280 nm. A molar extinction coefficient for AF488 of
(1)
where γ0 is the surface tension of the clean air−liquid interface (72.0 mN/m at 25 °C for water), and γ is the actual surface tension of the solution due to the adsorption of a surface-active material (e.g., mAbs). Surface pressure represents the reduction in the surface free energy and can be influenced by multiple properties of an interfacially adsorbed mAb film. For example, the surface area per adsorbed molecule, the solubility of the molecules, and the interactions of interfacial molecules with 3185
DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
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Surface Pressure. A KRÜ SS K100 force tensiometer provided surface tension measurements for the mAb solutions using a roughened platinum Wilhelmy plate. The chamber of the tensiometer was humidified to at least 70% relative humidity to limit evaporation. All measurements were performed at room temperature (22−24 °C). Surface pressure for each unlabeled mAb was measured at solution conditions identical to those used in the agitation stress study. Three milliliters of the mAb sample was placed into the tensiometer sample holder via reverse pipetting to prevent the introduction of any bubbles onto the air−liquid interface. Immediately after sample deposition, the tensiometer lowered the Wilhelmy plate into the interface and automatically detected the location of the interface. The surface pressure evolution was monitored for 30 min with measurements recorded every 0.5 s. Between each sample, the Wilhelmy plate was rinsed thoroughly with ultrapure water and then subjected to a flame from a butane torch to remove any contaminants. The tensiometer sample holder was extensively cleaned using acetone and ultrapure water. Periodic surface pressure measurements of ultrapure water ensured cleanliness of the tensiometer. The surface pressure for each mAb was measured three times in independent experiments. To characterize the initial increase in surface pressure for each mAb, the first 5 s of data (11 total points) were fit using a second-order polynomial, and the value of the derivative at time zero was calculated using the best-fit polynomial. Surface Excess. Surface excess, Γ, represents the amount of solute at an interface in excess of that which would be present if the bulk concentration extended all the way to the interface and represents the two-dimensional concentration of adsorbed films. The surface excess of each mAb was measured using confocal microscopy at room temperature. A Leica TCS SP5 inverted laser scanning microscope equipped with a 20×, 0.7 N.A. water-immersion objective provided the optical sectioning necessary to calculate surface excess. The air−liquid interface was formed in an eight-well sample holder adhered to a 170-μm thick, 3 in × 1 in borosilicate glass coverslip (ibidi; catalog numbers 80828 and 10812). To prevent evaporation during imaging, a wetted piece of filter paper was inserted into the sample holder cover. A hydrophilic glass surface ensured proper spreading of a thin film of mAb solution. Prior to use, the glass coverslips were soaked in a nonionic detergent solution overnight, rinsed with ultrapure water, dried, and subjected to a UV-ozone cleaner for 30 min to achieve a hydrophilic glass surface. The same solution conditions used for the agitation stress study were used for surface excess measurements. A volume of 15 μL of the AF488-mAb solution was deposited onto the glass surface of the eight-well slide, which produced an approximately 150-μm thick liquid film in the 1 cm2 well. To measure surface excess, a z-stack was acquired through the air−liquid interface from 12 μm below to 12 μm above the interface at 0.25 μm steps (excitation at 488 nm, detection at 510−600 nm). The average fluorescence intensity of each image slice was calculated and plotted versus axial location of the image slice. Convolving the measured point-spread function of the microscope with a two-box model for the distribution of mAb across the air−liquid interface yielded a theoretical fit to the observed data (Figure S1, Supporting Information). A custom Python script varied the fluorescence of the interfacial layer and the bulk fluorescence to minimize the sum of squared error between the measured and theoretical axial intensity
71 000 M−1 cm−1, mAb-specific extinction coefficients, and the A280/A494 correction factor of 0.11 for AF488 were all used to determine the degree of labeling. All mAbs had a degree of labeling of 0.6−0.7 mol AF488:mol mAb. Less than 10% free AF488 remained in the AF488-mAb stock solutions as measured by comparing the area of the late-eluting free dye peak to the total area of all peaks detected at 494 nm using SEC. Agitation Stress Study. A small-scale agitation study was performed in glass vials on an orbital shaker under high stress conditions to identify mAbs at risk for agitation-induced aggregation. mAbs were first diluted to 10 mg/mL in a low ionic strength buffer solution at pH 5.5. To approximate a dilution within the normal range used during IV bag compounding, a further 10-fold dilution into 0.9% NaCl was performed to obtain 10 mL of each mAb at 1 mg/mL. Each 10 mL sample was split between two 15-cc glass vials (5 mL in each vial) to ensure identical concentration in both agitated and nonagitated samples. The vials were stoppered, crimped, and warmed for 1 h in a 30 °C incubator. One vial of each mAb was then fixed horizontally on the platform of a Thermo Scientific MaxQ 4000 orbital shaker (0.75″ orbit) and agitated for 15 min at 170 rpm and 30 °C. Immediately following agitation, images of both the agitated and nonagitated controls were taken using a digital camera, and then further quantitative assays for aggregation were performed. Two independent agitation experiments of all 16 mAbs were performed to ensure reproducibility. SEC. Prior to soluble aggregate analysis via SEC, insoluble aggregates were removed from the sample by centrifugation at 16 000g for 10 min. A 35 min isocratic elution at 0.5 mL using the same mobile phase, column, and instrument as mentioned earlier allowed for quantification of relative amounts of monomer, dimer, and larger oligomers. The change in soluble aggregates was then determined by comparing agitated and nonagitated samples, with a change greater than 0.2% of total mAb indicating substantial aggregation. Turbidity. As a measure of insoluble aggregation, turbidity of the agitated and nonagitated samples was measured within 1 h postagitation using UV absorbance at an average wavelength of 350 nm (340−360 nm, 5 nm increments) using a 1 cm path length quartz cuvette. Immediately prior to measurement, the sample was gently inverted multiple times to resuspend insoluble aggregates. An increase in turbidity postagitation of more than 0.05 AU constituted substantial aggregation. Soluble Concentration Change. The decrease in soluble mAb concentration postagitation was used as an additional measure of insoluble aggregation. Insoluble mAb aggregates were first removed via centrifugation at 16 000g for 10 min, and then the supernatant was injected onto an HPLC without any column present. A 3 min isocratic elution at 0.5 mL/min using the same SEC mobile phase detailed previously ensured complete elution of mAb. The integrated area of four replicate injections, detected at 280 nm, provided the relative concentration change postagitation compared to the nonagitated control. As mentioned previously, the agitated and nonagitated samples were split from a single sample preparation to ensure identical starting concentrations. The integrated areas of the replicate injections had an average standard deviation of 0.5%. Assuming an error of two standard deviations for a concentration measurement and propagating this error, a decrease in soluble concentration of more than 1.5% indicated a substantial decrease due to aggregation. 3186
DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
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Figure 1. Images of the solution in each glass vial acquired immediately after the agitation stress study. The top set of images in each row represents the nonagitated controls, whereas the bottom set of images represents the same mAb that underwent 15 min of agitation on an orbital shaker. Asterisks and bold lettering indicate samples identified as containing aggregates upon visual inspection after agitation. The large particles formed in mAb-2 were particularly different than the aggregation observed in other mAb samples. Red text represents mAbs that demonstrated a high agitationinduced aggregation risk, whereas the dark cyan text represents low-risk mAbs.
(Figures 1 and 2). mAbs identified as having a high risk for agitation-induced aggregation exceeded the aggregation thresholds (see Experimental Section) in at least two of the three quantitative assays used: soluble mAb by protein concentration, turbidity, and soluble aggregates by SEC (Figure 2). Conversely, those categorized as exhibiting a low risk for aggregation did not exceed the threshold values for any of the assays performed. In addition, a qualitative visual inspection of the agitated and nonagitated vials confirmed the quantitative assays (Figure 1). Of particular note, the decrease in concentration of soluble mAb-1 after agitation and the increase in turbidity were double that of the next highest values observed, and the increase in soluble aggregates in mAb-1 was more than four-times the next highest increase. Overall, the panel of mAbs examined spanned a wide range of aggregation propensity and was therefore an appropriate set of molecules to use to relate interfacial properties to agitation stress susceptibility at an air−water interface. The agitation stress we employed resulted in both insoluble and soluble aggregates. mAbs that showed a substantial increase in soluble aggregates by SEC (Figure 2C) always had a corresponding increase in insoluble aggregates. However, mAb4 and mAb-5 formed substantial amounts of insoluble aggregates without an increase in soluble aggregates (compare Figure 2, panels A and B to panel C). Also, the increase in turbidity due to agitation stress directly correlated to a decrease in the soluble mAb concentration as expected during the formation of insoluble aggregates (Figure 2A,B). An exception to this correlation was mAb-2, which had a modest increase in turbidity but a large decrease in concentration. Agitation of mAb-2 resulted in the formation of large, visibly distinguishable particles (Figure 1). Although these particles contained a large mass of mAb, the suspension had a low measured turbidity due to it having a small number of larger particles in the scattering volume.27 Because of the discrepancy between turbidity and soluble protein loss for mAb-2, as well as the inability of SEC to assay insoluble aggregates, the best quantitative measure of agitation-induced aggregation for the panel of 16 mAbs was the decrease in soluble mAb concentration (Figure 2A).
profiles. The known mAb bulk concentration of 1 mg/mL was then used to calculate the absolute surface excess for every zstack acquired.24 Each surface excess value was determined from three independent measurements. The low degree of labeling with the AF488 fluorophore did not substantially affect the surface activity of the mAbs, as determined by measuring the surface excess of different ratios of labeled-to-unlabeled mAb (Figure S2, Supporting Information). Measured and Theoretical Hydrophobicities. The relative hydrophobicity of each mAb in its native conformation was assayed using hydrophobic interaction chromatography (HIC).25 HIC was performed using an Agilent 1100/1200 series HPLC equipped with a diode array detector and a Tosoh TSKgel Ether-5PW column (catalog number 08641). Mobile phase A consisted of 1.5 M (NH4)2SO4 and 25 mM NaOAc (pH 5.5), and mobile phase B consisted of 25 mM NaOAc (pH 5.5). Each mAb was diluted to 1 mg/mL prior to analysis and eluted at 0.8 mL/min using a multistep gradient: 0−100% B over 60 min, hold at 100% B for 10 min, and then return to 0% B and hold for 10 min. The hydrophobicities of the mAbs were compared to each other using relative hydrophobicity units (rHU) determined from retention times. Values of 5 and 95 rHU were assigned to two Genentech mAbs that have short and long retention times, respectively. A linear relationship between retention time and rHU was then determined using this two-point calibration. The measured relative hydrophobicity was also compared for two mAbs between the AF488-labeled and unlabeled molecules. The fluorophore did not substantially change the hydrophobicity of the mAbs (Figure S2, Supporting Information). In addition to the measured relative hydrophobicity of each mAb in the panel studied, a theoretical hydrophobicity was calculated using the primary sequence of the mAbs based on the methods of Bigelow.26
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RESULTS Agitation Stress Study. Out of a panel of 16 mAbs, seven high-risk antibodies were identified based on an agitationinduced aggregation study with no protective surfactant present 3187
DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
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Figure 3. Risk of agitation-induced aggregation correlated strongly with initial increase in surface pressure upon adsorption of the mAb to the air−water interface. (A) Surface pressure, Π, adsorption isotherms for all mAbs over the entire 30 min adsorption. High-risk mAbs are indicated in red, and low-risk mAbs are indicated in dark cyan. (B) Initial 1 min of surface pressure adsorption isotherms for all mAbs. The initial 5 s of data were used to calculate the slope at time zero. (C) Correlation between the decrease in soluble mAb concentration (due to insoluble aggregation) and the initial rate of increase in surface pressure. Red, solid points represent mAbs with a high risk for agitation-induced aggregation, and dark cyan, open points represent low-risk mAbs. The Pearson’s correlation coefficient, rp, is also given. All plotted surface pressure data represent the averages of three independent measurements, while the decrease in soluble mAb concentration data represents the averages from two independent agitation studies.
Figure 2. Determination of agitation-induced aggregation risk for a panel of 16 mAbs. (A) Percent decrease in concentration of the soluble fraction of mAb present in the supernatant after insoluble aggregates were removed via centrifugation. (B) Turbidity difference of agitated samples compared to nonagitated controls as measured by the absorbance at 350 nm without any centrifugation. (C) Change in percentage of soluble aggregates postagitation as measured using SEC. A negative value represents a lower amount of soluble aggregate present postagitation compared to the starting material. In all panels, dashed lines indicate the threshold for identifying substantial aggregation. Below panel C, agitated samples that appeared aggregated upon visual inspection are indicated with a plus sign (Figure 1). Red bars and symbols represent mAbs that demonstrated a high agitationinduced aggregation risk based on this assessment, whereas the dark cyan bars and symbols represent low-risk mAbs. Error bars represent the standard deviation calculated from two independent agitation studies.
ordering of the high-risk molecules (Figure 3C). A clear cutoff around 1.5 mN/m/min separated all the high-risk molecules from the low-risk molecules, with mAb-14 as the only false positive (Figure 3C). This cutoff essentially separated those mAbs that had a lag in measurable surface pressure increase upon being exposed to a fresh air−water interface from those that immediately increased in surface pressure (Figure 3B). mAbs with a lag had a low aggregation propensity, while those without a lag had a high aggregation propensity. In addition, the magnitude of the initial rate of surface pressure increase correlated with the severity of agitation-induced protein aggregation, suggesting that the rate of surface pressure increase may be used in a semiquantitative manner to predict the interfacially mediated aggregation propensity of an antibody. It should be noted that surface pressure magnitudes will depend on a variety of parameters, so comparison of different molecules should be performed using identical experimental conditions, as was done here. The one false positive identified using the surface pressure assay, mAb-14, also exhibited a unique surface pressure adsorption curve compared to the other mAbs. mAb-14 adsorbed to the highest overall surface pressure of the 16 molecules examined in this study (Figure 3A). In addition, the shape of the adsorption curve differed from the other mAbs, with a linear rate of increase during the first minute (Figure 3B). This differs from the initial adsorption of the high-risk mAbs, which showed a rapid increase in surface pressure that
Initial Increase in Surface Pressure Predicts Aggregation. Surface pressure generally represents the propensity of an amphiphilic molecule to adsorb to the air−liquid interface, with a higher surface pressure indicating a more surface-active molecule. Figure 3, panels A and B show the surface pressure adsorption isotherms for all 16 mAbs investigated. Importantly, all the high-risk mAbs exhibited an immediate and rapid increase in surface pressure upon being exposed to a fresh air− liquid interface. In contrast, all but one of the low-risk mAbs had a lag phase before a measurable surface pressure developed, as seen when the initial surface pressure change upon adsorption was observed (Figure 3B). By fitting the slope of the curves shown in Figure 3, panel B, we discovered that the initial rate of surface pressure increase was the best predictor of agitation-induced protein aggregation for the 16 mAbs (for details of the fitting, see Figure S3 and Table S1, Supporting Information). The decrease in soluble mAb concentration postagitation correlated very strongly (rp = 0.90) with the initial rate of surface pressure increase and enabled both the identification of high- and low-risk mAbs as well as the rank 3188
DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
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Molecular Pharmaceutics quickly slowed, suggesting unique interfacial properties for mAb-14. mAb-14, referred to as mAb1 by Liu and co-workers, was shown to self-associate through strong, reversible electrostatic interactions that resulted in unusually high solution viscosities.28 Such reversible interactions may also occur at the interface and would result in the high surface pressure observed here without inducing aggregation. To quantify the difference in shape between the curve for mAb-14 and the curves for the high-risk mAbs, the second derivative of the initial 5 s of the surface pressure adsorption curves was calculated (Table S1, Supporting Information). For the high-risk mAbs, the average value of the second derivative was −90 mN/m/min2, whereas for the low-risk mAbs, the average value was −10 mN/m/min2. The second derivative for mAb-14 was −10 mN/m/min2 and represented the lack of curvature in the initial surface pressure adsorption isotherm, which is a unique characteristic of the lowrisk mAbs. Aggregation Risk Not Well-Predicted by Other mAb Properties. Although the initial rate of surface pressure increase strongly correlated with interfacial mAb aggregation, the pseudoequilibrium surface pressure value at longer times did not accurately identify high-risk antibodies. Only a weak positive relationship existed between the aggregation propensity and the surface pressure of the mAb films at 20 min (Figure 4A). In addition, using the surface pressure at 20 min to identify all high-risk mAbs resulted in five false positives, an
unacceptable error rate. Surface pressure values from other times, between 5 and 30 min, also performed poorly at predicting aggregation propensity of the mAb panel. Beyond 5 min, the surface pressure increased at approximately the same rate for all the mAbs (Figure 3A), suggesting that the relative rank order in surface pressure values at 20 min represents the rank order in equilibrium surface pressure values for the various mAbs. The surface excess of mAb (i.e., the surface concentration) at the air−liquid interface also did not serve as an accurate predictor for agitation-induced protein aggregation. Aggregation propensity did moderately correlate with surface excess (Figure 4B), but many of the mAbs had surface excess values of 300−500 ng/cm2, with both high- and low-risk antibodies in this range. This would make it very difficult to identify a cutoff value to accurately identify high-risk mAbs with an acceptable number of false positives. The weak correlation between surface excess and interfacial aggregation propensity indicated that the amount of antibody adsorbed to the air−liquid interface did not directly determine the extent of aggregation during the agitation stress study. The surface excess values determined for the mAbs here using confocal microscopy agreed well with the approximately 500 ng/cm2 surface excess of a mix of human IgG at the air−liquid interface determined using a radiotracer technique.29 Hydrophobicity is generally considered a major contributor to protein aggregation, with hydrophobic proteins having a higher propensity to aggregate in bulk solutions.30,31 However, neither a measured surface hydrophobicity determined by HIC nor a calculated hydrophobicity based on primary sequence accurately predicted agitation-induced aggregation risk for the panel of 16 mAbs we studied. Calculated hydrophobicity demonstrated no correlation with aggregation risk (Figure 4D). Measured hydrophobicity, more representative of the exposed surface hydrophobicity of the mAbs, did correlate moderately with aggregation risk. However, use of measured hydrophobicity to identify all the high-risk mAbs resulted in three false positives. Overall, equilibrium surface pressure, equilibrium surface excess, and hydrophobicity of the mAbs did not predict aggregation risk, and all identified numerous false positives when choosing thresholds to correctly identify the group of high-risk mAbs. The inclusion of multiple interfacial and solution properties of the mAbs (e.g., surface pressure, surface excess, hydrophobicity, charge, melt temperature, and diffusion interaction parameter) in a predictive model of agitation-induced protein aggregation did not substantially improve upon the prediction provided by the initial rate of surface pressure increase as a single factor. Surface Excess Does Not Determine Surface Pressure of mAb Film. The surface pressure of the mAb films that we studied was likely determined primarily by the magnitude of self-interaction between surface-adsorbed mAb molecules and not the interfacial concentration of the antibodies. For example, the surface excesses of mAb-3 and mAb-13 reached equilibrium within 1 min of adsorption (Figure 5A), whereas the surface pressure for both these mAbs, and all others tested, continuously increased over the entire 30 min film evolution. Of particular note, the lag in surface pressure increase observed for mAb-13 did not correspond to a slow increase in surface excess. Therefore, the surface pressure lag did not indicate a lack of mAb adsorbed to the interface but instead may have been the result of a lack of measurable interactions between adsorbed antibody molecules. Across all 16 mAbs investigated
Figure 4. Agitation-induced aggregation risk did not correlate strongly with other mAb properties. The correlations are shown between the decrease in soluble mAb concentration and (A) the surface pressure measured at 20 min, Π20 min, (B) the equilibrium surface excess, Γequi, (C) the relative hydrophobicity of the mAbs measured using HIC, and (D) a calculated hydrophobicity of the mAbs based on their primary sequences. In all panels, red, solid points represent high-risk mAbs, and dark cyan, open points represent low-risk mAbs. 3189
DOI: 10.1021/acs.molpharmaceut.5b00089 Mol. Pharmaceutics 2015, 12, 3184−3193
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Molecular Pharmaceutics
Assuming the projected interfacial area of each antibody is composed of three identical ellipses, the maximum packing fraction on the interface would be π/(12)1/2 ≈ 0.907.34 Combining this maximum excluded area with eq 3 yields a maximum surface pressure of 1.46 mN/m, assuming the maximum observed surface excess of ∼800 ng/cm2, which still does not account for the observed surface pressure increases of more than 5 mN/m (Figure 3A). To correct for effects not considered in eqs 2 and 3, researchers have developed a variety of equations of state to explain the real adsorption behavior of globular proteins.35−38 These models allow for multiple adsorbed states of a molecule represented by differing molecular areas as well as an interaction parameter to account for the nonideality of mixing of the molecules at the interface. We hypothesized that attractive protein−protein interactions between the interfacially adsorbed molecules accounted for the large surface pressure of the mAb films we studied. Because of the tight packing of the interfacial mAb monolayer, as determined from the magnitude of the surface excesses of the mAbs, the molecular areas of the adsorbed molecules likely did not change substantially once the film reached an equilibrium surface excess. This equilibrium was reached within 1 min, as demonstrated by two representative mAbs (mAb-3 and mAb13), whereas the surface pressure continued to increase while at this equilibrium surface excess (Figure 5A). The dramatic increase in surface pressure may have resulted from a slow rearrangement or conformational change of the adsorbed mAb molecules that occurred over a time scale of several minutes to an hour. Such a change could greatly affect protein−protein interactions at the interface, thereby leading to a large increase in surface pressure at constant surface excess. A similar phenomenon was reported for the adsorption of lysozyme to the air−water interface where the surface pressure increased on a substantially slower time scale than the surface excess increased.22 Although both attractive and repulsive interactions may lead to an increase in surface pressure, the measured interactions here resulted in protein aggregation, suggesting they were attractive in nature. These attractive interactions were likely hydrophobically driven possibly through favorable alignment of hydrophobic moieties on the mAbs at the interface or through partial unfolding. Partial unfolding could also expose buried, unpaired cysteines in some antibodies that can lead to covalently linked aggregates.39 Gross conformational changes, such as denaturation, likely did not account for the surface pressure increase in the mAb films studied here since that would have required a substantial increase in the interfacial area occupied by each mAb causing a measurable difference in surface excess over time, which was not observed (Figure 5A). Instead, if surface pressure here was predominantly a measure of attractive interfacial protein− protein interactions in the mAb film at the air−liquid interface, then the rate of change in surface pressure upon adsorption measured how quickly adsorbed molecules began to selfassociate. Others have attributed a lag in the increase in surface pressure upon adsorption of albumin to the air−water interface to a lack of interfacial protein−protein interactions, which is consistent with our hypothesis.33,40 In this study, the adsorbed mAb molecules likely only persisted on the air−liquid interface for a short time before the interface was destroyed and recreated; thus, the rate of self-association (measured as an increase in surface pressure) determined the extent of aggregation (Figure 3C). A mAb that shows little aggregation during agitation with a short average surface age (i.e., seconds)
Figure 5. Differences in surface pressure for the various mAbs are not dominated by the quantity of mAb adsorbed to the air−liquid interface. (A) For two mAbs, the time evolution of surface excess, Γ, was compared to the evolution of surface pressure, Π, under identical conditions. Surface excess quickly equilibrated for both, albeit at different levels. However, the surface pressure of mAb-3 quickly increased upon adsorption to the interface, whereas mAb-13 had a lag phase before a measurable surface pressure developed. The solid lines indicate the surface pressure of the film and the open points indicate the surface excess. (B) The surface pressure measured at 20 min, Π20 min, did not correlate with the equilibrium surface excess, Γequi, for the 16 mAbs tested. All plotted values represent the averages of three independent measurements.
here, only 12% of the variance in the surface pressure was explained by differences in surface excess (Figure 5B; r2 = 0.12), demonstrating that the magnitude of surface pressure was not dominated by the interfacial concentration of the mAbs.
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DISCUSSION Our results suggest that the initial rate of surface pressure increase, which correlated strongly with the extent of agitationinduced aggregation of the mAb panel used in this study, represents the rate at which strong, attractive interactions develop between molecules adsorbed to the air−liquid interface. Under ideal, noninteracting conditions, surface pressure should depend directly on the surface excess of adsorbed molecules. Analogous to the three-dimensional ideal gas law, in two dimensions: RT = Γ̂RT (2) A where R is the gas constant, T is temperature, A is the mean molecular area, and Γ̂ is the molar surface excess. However, for a surface excess of ∼800 ng/cm2 (5.5 × 10−8 mol/m2), the highest observed for the mAbs studied here (Figure 4B), eq 2 gives a surface pressure of only 0.14 mN/m at 25 °C. This surface pressure is within the measurement variability of the Wilhelmy plate method and much less than the surface pressures observed for the mAb films (Figure 3A). This clearly indicates that the mAb films do not behave ideally, which would only be expected for dilute films of noninteracting protein molecules. On the basis of typical antibody sizes, a well-packed monolayer contains 200−550 ng/cm2 depending on molecular orientation,32 suggesting the mAbs studied here (Figure 4B, surface excesses of 300−800 ng/cm2) adsorbed in well-packed monolayers potentially with a second diffuse adsorbed layer. To account for the dense packing of the mAb monolayer, eq 2 can be extended to account for the excluded surface area of the molecules, α:33 Π=
⎛1 ⎞ Π⎜ − α⎟ = RT ⎝ Γ̂ ⎠
(3) 3190
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results in significant dilution of the surfactant, but a minimal concentration would still be present, unlike in the stress model used here in which no surfactant was added. While in-use IV bag studies serve a critical role in both early- and late-stage development of biopharmaceuticals, the approach presented here is well-suited for molecular assessment where the goal is to identify risk factors for the mAb of interest and to decide between candidate molecules. Even though simply identifying high- and low-risk molecules would have been sufficient, the rate of surface pressure increase also semiquantitatively predicted the extent of aggregation (Figure 3C). The kinetics of adsorption greatly influence the interfacial susceptibility of the mAbs during agitation stress, and those most prone to aggregation likely would need higher concentrations of a surfactant-like polysorbate 20 to protect against such aggregation through competitive adsorption of the surfactant to the air−liquid interface. Initial surface pressure change offers several practical advantages as a screening tool for molecular assessment. Using a Wilhelmy plate tensiometer like the instrument we employed, a series of surface pressure measurements requires approximately 10 mg of protein, a reasonable quantity to use during early development. If using a pendant drop tensiometer, this sample requirement would be reduced to less than 1 mg, enabling the screening for agitation-induced aggregation risk even before entering into development. In addition, the surface pressure measurements required no special sample preparation (e.g., fluorescent labeling, enzymatic digestion), and, if only concerned with the initial rate of surface pressure increase, can be completed in less than an hour. Although we have demonstrated the predictive power of the surface pressure of mAb therapeutics in assessing their aggregation risk, much remains to be learned by further investigation of the interfacial phenomena of biotherapeutics. For example, understanding the mechanistic differences between various protective surfactants, examining protein aggregation at oil−water and solid−water interfaces, and exploring the effects that nonsurface active excipients have on the interfacial properties of protein therapeutics are areas relevant to biotherapeutic development that would benefit from further detailed interfacial characterization.
may still aggregate if the surface age could be artificially lengthened to many minutes or hours holding all else constant. Previous work demonstrated that the interfacially mediated aggregation of a mAb depended on the rate of interface turnover and that an intermediate turnover rate yielded the highest overall aggregation due to an optimum balance between aging of the mAb on the interface and amount of interface created in a given time period.19 In addition to mAbs, the length of interfacial exposure is likely an important parameter in aggregation of other proteins. For example, amyloid beta, an amphiphilic peptide implicated in Alzheimer’s disease, showed a considerable lag in surface pressure increase41 but had a high propensity to aggregate at an air−liquid interface during prolonged exposure.42 This reiterates the importance of examining the kinetics of protein adsorption when considering agitation-induced aggregation, where the air−liquid interface persists for only a short time before being destroyed and recreated. mAb hydrophobicity alone did not predict interfacially mediated aggregation well; however, there was a positive relationship between measured relative hydrophobicity and equilibrium surface excess across the panel of mAbs investigated (rp = 0.65; plot not shown). This correlation is consistent with a previous study where increased hydrophobicity led to greater surface excess for a chemically modified antibody adsorbing to latex spheres.43 Although an individual mAb molecule may be hydrophobic, its orientation on the interface may not allow for attractive interactions with neighboring molecules that lead to aggregation. In addition, a mAb could have a small but highly hydrophobic patch that imparts the necessary amphiphilicity to promote the adsorption of a large interfacial concentration of antibody but not to promote aggregation with other molecules at the interface. In contrast to measured hydrophobicity, the calculated hydrophobicities of the mAbs based solely on their primary sequences demonstrated no relationship to either aggregation risk (Figure 4D) or surface excess. One would only expect primary sequence hydrophobicity to be a good predictor of aggregation and surface properties if the mAb lost a majority of its secondary and tertiary structure upon adsorption to the air− liquid interface. Although the mAbs may partially unfold at the air−liquid interface, they likely retain some structure since the protein surface coverages in this study would not allow for sufficient interfacial area per molecule for complete denaturation to occur.38,44 The inter- and intrachain disulfide bonds in the mAbs should also maintain protein tertiary structure at the interface. Even in the case of a protein without much secondary or tertiary structure, additional properties, such as the relative ratio of hydrophobic to hydrophilic amino acids and the distribution of hydrophobic amino acids among the hydrophilic amino acids, are expected to play a significant role in determining solubility and aggregation. Instead of a bulk or theoretical measurement of hydrophobicity, an in situ measurement of film hydrophobicity could yield further insight into the relationship between hydrophobicity within the context of the air−liquid interface and its relationship to aggregation. The goal of our study was to assess the viability of measuring interfacial properties to screen for mAbs at high risk for agitation-induced aggregation. The agitation conditions we used were not intended to replicate in-use conditions. Under a typical in-use agitation study, drug product containing a nonionic surfactant such as polysorbate 20 would be diluted into IV bags in a manner similar to that in the clinic. This
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00089. Calculation of antibody surface excess at the air−liquid interface using confocal microscopy; the effect the fluorophore has on antibody surface activity and hydrophobicity; the first 5 s of surface pressure data for all the antibodies studied; for all antibodies, the decrease in concentration post agitation as well as the first and second derivatives of the surface pressure data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: (650) 467-7427. Notes
The authors declare the following competing financial interest(s): The authors, Ian C. Shieh and Ankit R. Patel, are both employed by Genentech, Inc. 3191
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ACKNOWLEDGMENTS We thank Timothy Spirakes, Brian Hosken, and Boyan Zhang for developing the relative hydrophobicity HIC method and Danielle Leiske for many productive discussions. We also thank our colleagues in the Early Stage and Late Stage Pharmaceutical Development departments at Genentech for providing antibodies for this work.
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