Effect of Excipients on Liquid–Liquid Phase Separation and

Jan 12, 2016 - The pH of all the above solutions was within ±0.1 of the target values as ... Supernatant was further diluted down to 1 mg/mL and dire...
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Effect of Excipients on Liquid−Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions Ashlesha S. Raut* and Devendra S. Kalonia* Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, Connecticut 06269, United States ABSTRACT: Liquid−liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature. In this study, the effect of four excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl beta-cyclodextrin (HPβCD)) on liquid−liquid phase separation and aggregation in a dual variable domain immunoglobulin protein solution was investigated. Sucrose suppressed both LLPS and aggregation, Tween 80 had no effect on either, and PEG 400 increased LLPS and aggregation. Attractive protein−protein interactions and liquid−liquid phase separation decreased with increasing concentration of HPβCD, indicating its specific binding to the protein. However, HPβCD had no effect on the formation of soluble aggregates and fragments in this study. LLPS and aggregation are highly temperature dependent; at low temperature protein exhibits LLPS, at high temperature protein exhibits aggregation, and at an intermediate temperature both phenomena occur simultaneously depending on the solution conditions. KEYWORDS: liquid−liquid phase separation, aggregation, excipients, protein−protein interactions, physical stability, protein formulation, sucrose, PEG, hydroxypropyl beta-cyclodextrin (HPβCD)

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INTRODUCTION The aim of formulation development of protein therapeutics is to produce efficacious products wherein the protein remains in the solution and maintains stability across its shelf life. Phase separation and aggregate formation in protein solutions can compromise physical stability of the formulation.1,2 Protein solutions can exhibit solid−liquid phase separation resulting in the formation of either crystals or amorphous precipitate or a metastable liquid−liquid phase separation into a protein-rich and a protein-poor phase.3−5 In either case, protein retains its native structure in the formulation, and the decreased solubility is associated with the change in the chemical potential of the protein in solution or increased intermolecular interactions between the protein molecules. Aggregation on the other hand is associated with native and/or non-native species and can be reversible or irreversible in nature.6 Aggregates can result in reduced efficacy of the product and can elicit immunogenic response.7 Aggregation of the therapeutic proteins has been extensively reported in the literature, and readers are referred to some excellent reviews on aggregation pathways (physical and chemical)8,9 and kinetics10 in solution, general terminologies associated with classification of aggregates,11 and assessment of aggregates12−14 in protein solutions.15,16 © 2016 American Chemical Society

Excipients play a significant role in maintaining the physical stability of protein formulations.17,18 Depending on the concentration and nature, certain excipients can exert dual effect, e.g., preferentially excluded cosolutes increase the conformational stability of proteins in solution, while they can also promote phase separation (or reduced solubility) if the increase in the chemical potential of the protein in solution phase exceeds that in the solid phase.19 Excipients exert their stabilizing effect by modulating protein−protein interactions in solution or by changing conformational stability. Sugars, polyols, polymers, salts, surfactants, amino acids, and their derivatives are some of the excipients used in therapeutic protein formulations to improve physical stability of the protein solution.17,18 In this study, the effect of excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl β-cyclodextrin) on liquid−liquid phase separation (LLPS) and aggregation in DVD-Ig protein solution was investigated. PEG 400 is a macromolecular crowding agent and is a commonly used protein precipitant; Received: Revised: Accepted: Published: 774

September 2, 2015 January 8, 2016 January 12, 2016 January 12, 2016 DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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Molecular Pharmaceutics

filtered through 0.22 μm Millipore Durapore membrane filters, and then diluted down to the required concentrations with the same buffer (stock: 200 mM HPβCD, 50% PEG 400, 40% sucrose, 0.1% Tween 80). All antibody solutions were buffer exchanged using Millipore (Billerica, MA) Amicon Ultra centrifugation tubes with a molecular weight cutoff of 10 kDa obtained from Fisher Scientific. Concentrations of the samples were determined using a UV spectrophotometer with an extinction coefficient of 1.5 mL/mg cm−1 for DVD-Ig protein at 280 nm for 0.1% (w/v) IgG solutions. The pH of all the above solutions was within ±0.1 of the target values as measured by a Denver Instrument pH meter. Methods. Accelerated Stability Studies. DVD-Ig protein samples with a concentration of 33 mg/mL were prepared in histidine buffer, pH 6.6, 15 mM ionic strength, and appropriate concentration of excipients was maintained (15, 30, and 50 mM HPβCD, 10% sucrose, 10% PEG, 0.01% Tween). All the solutions were filtered through sterile 0.22 μm Millipore Durapore membrane filters before incubation. 150 μL samples for each condition (in duplicate) were filled in sterile Fischerbrand (Fischer Scientific) 0.5 mL microcentrifuge tubes with loop and O-ring and incubated at 4 °C in refrigerator and at 20 and 40 °C in isotemp oven (Fischer Scientific). At the set time points (7, 21, and 45 days), samples were brought to room temperature and thoroughly mixed with pipettes to homogenize the solution (in case phase separation occurred on standing at a set temperature). Appropriate amount of aliquot was withdrawn and diluted to around 2 mg/ mL and then centrifuged at 6708g for 5 min using an Eppendorf minispin (HA, Germany). Supernatant was further diluted down to 1 mg/mL and directly filtered through 0.22 μm syringe filters into HPLC vials. At the 45 day time point, samples were also analyzed for structural changes by second derivative fluorescence spectroscopy (0.2 mg/mL concentration at all conditions was analyzed; other steps are similar to that reported for intrinsic fluorescence). A third set of samples were stored in Eppendorf tubes at 4 °C, which were analyzed for concentration of protein in protein-rich and protein-poor phase (to assess phase separation). HPLC Size Exclusion Chromatography. Analysis of the stability samples was conducted using an SEC-HPLC system with an inline UV detector set at 280 nm. A TSK G3000 SWXL gel filtration column (Tosoh Biosciences) was used for separation. 100 mM sodium phosphate buffer at pH 7 containing 200 mM sodium sulfate was used as mobile phase for elution. Flow rate was maintained at 0.3 mL/min, and 10 μL of sample volume was injected into the system. HPLC analysis was also performed for initial samples before subjecting to stress conditions to determine monomer and aggregate content (labeled as t0). Samples were collected and analyzed at 7 days, 21 days, and 45 days. Chromatograms obtained were analyzed for percent of monomer, soluble aggregates, and fragments using ChemStation software. Intrinsic Fluorescence. Fluorescence spectra for protein− excipient samples were acquired using a Photon Technology International (PTI) 814 spectrofluorometer, equipped with a turreted 4-position Peltier-controlled cell holder. DVD-Ig protein samples were prepared at a concentration of 0.2 mg/ mL at pH 6.6 in histidine buffer (15 mM ionic strength) keeping the excipient concentration constant. An excitation wavelength of 295 nm was used, and the emission scans were recorded from 310 to 450 nm with a step size of 2 nm and a 1 s

it enhances conformational stability of proteins in solution by the preferential exclusion mechanism.20 Extensive studies for PEG-induced LLPS are reported in the literature for therapeutic proteins including mAbs.21−24 Sucrose is a widely used protein stabilizing excipient against thermal denaturation, which is preferentially excluded, and exerts its effect by increasing the surface tension of water.25−28 Tween 80 is a surfactant which competes with the protein to adsorb at the air−water interface, thereby reducing its tendency to denature at the interface.29−31 Recently, β-cyclodextrin (β-CD) and its derivatives have been reported to successfully suppress aggregation in protein solutions.32 CDs are cyclical oligosaccharides consisting of (α1,4)-linked α-D-glucopyranose units with a hydrophilic exterior and a hydrophobic core. Solubility of unsubstituted β-CD is around 18.5 mg/mL in water, while solubility of hydroxypropyl β-cyclodextrin (HPβCD) and sulfobutyl ether β-cyclodextrin (SBβCD) is greater than 600 and 500 mg/mL in water, respectively, resulting in improved solubility of poorly soluble pharmaceutical drugs.33−35 HPβCDs have been reported to decrease aggregation in protein solutions by binding to or encapsulating the aromatic side chains (tryptophan, tyrosine, and phenylalanine) and histidine with its imidazole side chain, into its hydrophobic core.36−39 However, there are other mechanisms reported in the literature by which HPβCD reduces aggregate formation in solution. The degree of substitution of CD hydroxyl groups determines its surface activity. HPβCD has been reported to act similar to surfactants and is adsorbed at the air−water interface competing with the protein and hence reducing surface induced aggregation.39−42 HPβCD has also been hypothesized to be preferentially excluded from the surface of the protein due to its large size, resulting in protein hydration and preventing it from unfolding and hence reducing aggregation.32,36 Though many reports in the literature highlight the effectiveness of sucrose, Tween 80, and the potential of HPβCD in reducing aggregation of the proteins in solution, to the best of our knowledge, there are no studies on the effect of these excipients on liquid−liquid phase separation in DVD-Ig protein solution. The purpose of the current study was (i) to mechanistically investigate the effect of excipients on LLPS and aggregation in DVD-Ig protein and (ii) to understand the relationship between LLPS and aggregate formation in DVD-Ig protein solution in the presence of the excipients and as a function of temperature.

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MATERIALS AND METHODS Materials. All buffer reagents and chemicals used were of the highest purity grade and were obtained from commercial sources. Histidine base, histidine hydrochloride, and hydroxypropyl β-cyclodextrin (MW-1380) were purchased from Sigma (St. Louis, MO). Monobasic and dibasic sodium phosphate, sodium sulfate, sucrose, Tween 80, and polyethylene glycol 400 were purchased from Fisher Scientific (Fair Lawn, NJ). Dual variable domain antibody, DVD-Ig protein (pI ∼ 7.5; mol wt ∼ 200 kDa) as 85 mg/mL solution in 15 mM histidine buffer at pH 5.5, was supplied by Abbvie (Worcester, MA). Solutions were prepared with deionized water equivalent to Milli-Q. Phosphate and histidine buffers were used to maintain pH at 6.6. Ionic strength of 15 mM was maintained by selecting appropriate buffer strength without addition of any salts. All the excipient stock solutions were prepared in appropriate buffers, 775

DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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Molecular Pharmaceutics integration time. The excitation and emission slit width was set at 2 nm, and eight emission spectra were collected and averaged to obtain the final spectrum. Sample volume of 60 μL was placed in 3 mm path length quartz cuvettes, and measurements were made at 25 °C after equilibration for 2 min. The buffer peak was subtracted from each spectrum, and all the emission scans were normalized to 1.0 (FeliX32 software, PTI) before obtaining second order derivative spectra for each solution condition. Tcloud Measurements. Temperature studies were performed using a UV−vis spectrophotometer attached to a temperature control Peltier plate, where percent transmittance was recorded as temperature was lowered in steps from 35 to 3 °C. 60 μL samples were placed in 3 mm quartz cuvettes, and allowed to equilibrate at each temperature for 2 min before measurements were recorded. Onset of the liquid−liquid phase separation is characterized by a dramatic increase in solution turbidity; this temperature is marked as Tcloud. For the current study, the temperature at which percent transmittance is 70 is termed as Tcloud. Protein concentrations of 16 and 60 mg/mL were analyzed for HPβCD, while concentration of 16 mg/mL was analyzed for sucrose, PEG, and Tween 80. After addition of excipients to protein solutions, all the samples were allowed to equilibrate for 2 h and filtered directly into the cuvette before making the measurement. Dynamic Light Scattering (DLS). DLS studies were performed using a Malvern Instrument Zetasizer Nano Series at a wavelength of a 632.8 nm and at 173°. All the buffers and sample stock solutions were filtered through 0.22 μm Millipore Durapore membrane filters before making the dilution to required concentrations. Excipient concentration was kept constant, and protein concentration was varied from 0.25 to 10 mg/mL for each set of measurements in duplicate. The protein and excipient solutions were then centrifuged using an Eppendorf minispin minicentrifuge at 12110g for 5 min before every measurement. Effect of temperature (11 °C, 18 °C, and 25 °C) on kD in the presence (30 mM) and absence (0 mM) of HPβCD was also investigated. Light scattering measurements for other excipients were made at 25 ± 0.1 °C. Linear plot of Dm against concentration was used to calculate self-diffusion coefficient (Ds) and interaction parameter (kD) using following relation: Dm = Ds(1 + kDc)

c is the concentration of the protein (g/mL).

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Figure 1. (a) Percent soluble aggregates and (b) fragmentation observed for DVD-Ig stored at 4 °C in the presence and absence of excipients, at t = 0, 7, 21, and 45 days. Samples were analyzed at a concentration of 1 mg/mL. Percent aggregates were calculated by SEHPLC using ChemStation software. All solutions were prepared in pH 6.6 histidine buffer with ionic strength of 15 mM. Mobile phase: 100 mM sodium phosphate buffer at pH 7 containing 200 mM sodium sulfate. All samples were analyzed at 25 ± 1 °C in duplicate, plot represents mean value, and error bars represent range.

No change in percent aggregate or fragmentation was observed after samples were incubated for 45 days. Stability Studies at 20 °C. Percent aggregates and fragmentation for DVD-Ig protein after incubation at 20 °C are plotted in Figure 2. From Figure 2a, after 7 days, no change in aggregate content was observed in any of the samples (within experimental error). However, after 21 days, percent aggregates increased for DVD-Ig protein in the presence of PEG 400, which further increased for samples analyzed after 45 days. There was no change in aggregate content in the presence of other excipients. From Figure 2b, no fragmentation was observed at any of the conditions after incubation for 45 days. Stability Studies at 40 °C. Figures 3a and 3b show plots of percent aggregates and fragmentation, respectively, for DVD-Ig protein incubated with different excipients at 40 °C. From Figure 3a, percent aggregate shows an increase after 7 days compared to that analyzed at t = 0, and increased significantly after 21 and 45 days at all the conditions. HPβCD showed a concentration dependent increase in aggregate formation at 7 and 21 days, however, after 45 days, the amount of aggregates was almost similar for the protein in buffer and at all the concentrations of HPβCD studied. In the presence of PEG 400, percent aggregates increased significantly (by ∼12%) after 7 days compared to protein samples in the presence of other

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RESULTS Effect of Excipients on Aggregation. Formation of aggregates in a protein solution compromises the physical stability of the formulation and can also reduce its efficacy and safety. Determining or quantifying the amount of aggregates in solution in the presence of different excipients can help in optimizing the solution conditions to increase the shelf life of the product. Figures 1 through 3 represent the effect of different excipients on soluble aggregation (high molecular weight species and fragments) in DVD-Ig protein samples incubated at different temperatures over a period of 45 days. Stability Studies at 4 °C. Figure 1 shows plots of percent aggregates (Figure 1a) and fragmentation (Figure 1b) of DVDIg protein as a function of different excipients after 7, 21, and 45 days at 4 °C. Before the samples were subjected to stress conditions, a small amount of aggregate was present in the solution (t0 ∼ 1.5%) as analyzed with SEC-HPLC at time t = 0. 776

DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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Figure 3. (a) Percent soluble aggregates and (b) fragmentation observed for DVD-Ig incubated at 40 °C in the presence and absence of excipients, at t = 0, 7, 21, and 45 days. Samples were analyzed at a concentration of 1 mg/mL. Percent aggregates were calculated by SEHPLC using ChemStation software. All solutions were prepared in pH 6.6 histidine buffer with ionic strength of 15 mM. Mobile phase: 100 mM sodium phosphate buffer at pH 7 containing 200 mM sodium sulfate. All samples were analyzed at 25 ± 1 °C in duplicate, plot represents mean value, and error bars represent range.

Figure 2. (a) Percent soluble aggregates and (b) fragmentation observed for DVD-Ig stored at 20 °C in the presence and absence of excipients, at t = 0, 7, 21, and 45 days. Samples were analyzed at a concentration of 1 mg/mL. Percent aggregates were calculated by SEHPLC using ChemStation software. All solutions were prepared in pH 6.6 histidine buffer with ionic strength of 15 mM. Mobile phase: 100 mM sodium phosphate buffer at pH 7 containing 200 mM sodium sulfate. All samples were analyzed at 25 ± 1 °C in duplicate, plot represents mean value, and error bars represent range.

second derivative spectra did not show any change in the tertiary structure for samples at any of the conditions studied. Hydrophobic amino acids usually reside in the core of the protein (in both native and aggregated form) to reduce the free energy of the system, and very few hydrophobic amino acids are present on the protein surface. Small change in signal intensity due to minor structural perturbations (which may be reversible on diluting the sample) may not be sufficient to be detected by fluorescence spectroscopy for the samples that exhibited aggregation at high temperatures. Effect of Excipients on Liquid−Liquid Phase Separation. Liquid−liquid phase separation (LLPS) is a concern in protein formulation development as the protein solutions are generally stored at refrigerated conditions (4−8 °C) where they show a higher tendency to phase separate. LLPS in solution can be attributed to attractive protein−protein interactions (PPI).46−49 Excipients that decrease the interactions between the protein molecules will reduce their tendency to undergo LLPS in solution.49,50 A decrease in Tcloud or shift to lower temperatures indicates reduced attractive interactions and, hence, decreased LLPS in the solution. Tcloud was determined by measuring percent transmittance as solution temperature was lowered to qualitatively determine the effect of excipients on LLPS in DVD-Ig protein solution. PPI in the presence of

excipients. However, the rate of aggregate formation decreased from day 21 to day 45. Amounts of aggregates in the presence of Tween 80 are almost similar to buffer (absence of excipients). Sucrose showed increased thermal stability as percent aggregate content was lower than in buffer. Percent fragmentation (Figure 3b) for DVD-Ig protein showed a significant increase at all the conditions after 45 days. In the presence of HPβCD, percent fragmentation is similar to that in buffer, and there is no effect of increasing HPβCD concentration. Similarly, for Tween and sucrose, percent fragmentation is almost equal to that observed in the buffer at all the time points. However, for PEG, percent fragmentation increased significantly after 21 days and 45 days while aggregate formation remained nearly constant from 21 to 45 days. Structural Changes. Intrinsic fluorescence and second derivative fluorescence spectroscopy44,45 studies were performed for DVD-Ig protein in the presence and absence of excipients to determine tertiary structural changes after samples were incubated at 40 °C for 45 days (data not shown). Fluorescence spectra were also recorded for freshly prepared DVD-Ig protein samples at similar solution conditions. No shift in intensity or wavelength (blue shift or red shift) in the presence or absence of excipients was observed. Similarly, 777

DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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Molecular Pharmaceutics different excipients was further confirmed by well-established light scattering studies, where kD or interaction parameter was determined from diffusion coefficient measured using dynamic light scattering. A positive kD value indicates repulsive PPI, while a negative kD value indicates attractions between the protein molecules. All the negative kD values in the current study are outside the hydrodynamic range of 0 to −9 mL/g (small range where interactions are repulsive in nature, indicated by positive B2 values, but kD values are negative due to larger hydrodynamic contributions) and hence indicate attractions between the molecules. Figure 4 shows a plot of Tcloud on the primary axis and kD on the secondary axis against different excipients (10% PEG 400,

Figure 5. Plot of Tcloud (temperature where percent transmittance is 70) for DVD-Ig at pH 6.6 as a function of increasing concentration of HPβCD. Protein concentration of 16 mg/mL in phosphate and histidine buffers and protein concentration of 60 mg/mL in histidine buffer were analyzed. 200 mM stock solution of HPβCD was prepared in both histidine and phosphate buffer at pH 6.6 with ionic strength of 15 mM and then diluted down to required concentration using the same buffers. All solutions were analyzed in duplicate, plot represents mean value, and error bars represent range.

shows a plot of Tcloud for 16 mg/mL protein solution as a function of increasing HPβCD concentration (in histidine and phosphate buffers) and for 60 mg/mL protein concentrations (in histidine buffer) at pH 6.6, 15 mM ionic strength. In the absence of any excipients (only buffer), DVD-Ig protein exhibited high Tcloud in both the buffers. In histidine buffer, 60 mg/mL solution showed slightly higher Tcloud (22 °C) than 16 mg/mL solution (20 °C). The phase separation temperature was significantly higher for 16 mg/mL solutions in phosphate buffer (30.5 °C). Tcloud decreased with increasing concentration of HPβCD for DVD-Ig protein samples in both phosphate and histidine buffers. Although the buffer species affect Tcloud, the effect of HPβCD is independent of the buffer type. Figure 6 shows a plot of kD as a function of HPβCD concentration at 25 °C. In the absence of any excipients, DVD-

Figure 4. Plot of Tcloud (primary axis) for DVD-Ig at a concentration of 16 mg/mL determined from temperature studies and kD (secondary axis) determined from dynamic light scattering in the presence and absence of excipients. Stock solutions of PEG 400, sucrose, and Tween 80 were prepared in histidine buffer at pH 6.6 with ionic strength of 15 mM and then diluted appropriately to obtain final concentrations of 10% PEG, 10% sucrose, and 0.01% Tween. Tcloud is termed as the temperature where percent transmittance is 70. kD is obtained from slope/intercept of the linear plot of mutual diffusion coefficient (Dm) against protein concentration. Light scattering measurements were made at 25 ± 0.1 °C. All solutions were analyzed in duplicate, plot represents mean value, and error bars represent range. Error bars if not visible are smaller than the symbols used.

10% sucrose, and 0.01% Tween 80). The pH and ionic strength of the solutions were maintained constant at pH 6.6 (histidine buffer) and 15 mM, respectively. In the absence of any excipients, Tcloud for DVD-Ig protein is around 20 °C and kD shows a large negative value (−60 mL/g) indicating strong attractive interactions in the solution. In the presence of 10% PEG, Tcloud increases to ∼25.5 °C and kD becomes more negative (∼−68 mL/g), both indicating increased attractive interactions in the solution and reduced solution stability. Tween (0.01%) had no effect on Tcloud (∼19.5 °C) and kD (∼−61.5 mL/g) measured for DVD-Ig protein compared to buffer alone. The effect of a higher concentration of Tween (0.05%) on Tcloud and kD was also studied; no change in Tcloud or kD was observed (data not shown). Sucrose (10% w/v) shows a significant decrease in Tcloud (∼11 °C) of the solution as compared to DVD-Ig protein in buffer. Attractive interactions in solution also decrease as indicated by a less negative kD value (∼−35 mL/g). Tcloud and kD were also determined for DVD-Ig protein in the presence of increasing concentration of HPβCD. Figure 5

Figure 6. Plot of kD determined from dynamic light scattering as a function of concentration of HPβCD at pH 6.6 in histidine buffer with 15 mM ionic strength. kD is obtained from the slope/intercept of the linear plot of mutual diffusion coefficient (Dm) against protein concentration. All the solutions were analyzed at 25 ± 0.1 °C in duplicate, plot represents mean value, and error bars represent range. 778

DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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Molecular Pharmaceutics Ig protein shows a large negative kD indicating the presence of strong attractive PPI in solution. On increasing the HPβCD concentration from 5 mM up to 50 mM, kD becomes less negative. However, the decrease in Tcloud at 16 mg/mL protein concentration (Figure 5) on increasing the HPβCD concentration from 0 mM to 50 mM (20 °C in buffer to 6 °C in 50 mM HPβCD) is more substantial compared to the decrease in attractive interactions as indicated by kD which were determined in dilute solutions (kD = −60 mL/g in buffer and kD = −40 mL/g in 50 mM HPβCD).

analysis of the aggregate type (covalent or noncovalent) needs to be performed for further understanding. Surfactant. Tween 80 has no effect on either aggregation (Figures 1−3) or LLPS (Figure 4) in solution. The amount of aggregates and fragments in the presence of Tween is almost similar to that in buffer alone. Also, the Tcloud and kD values are similar to the values in buffer. Surfactants are amphiphilic molecules and adsorb/concentrate at the air−water interface, reducing the concentration in the bulk. Low concentration of Tween in its monomeric form is the possible reason that no effect on either phenomenon was observed. Though Tween 80 stabilizes protein against surface-induced aggregation, it has been reported to destabilize proteins against thermal degradation29,60 and is also known to induce chemical degradation in proteins via oxidation due to formation of peroxides.31,61 Specific Binding. Effect of increasing concentrations of HPβCD on LLPS and aggregation was investigated to get a better understanding of the mechanism by which it interacts with the protein (compete to adsorb at interface vs binding to hydrophobic groups). HPβCD does not affect aggregate formation or fragmentation in DVD-Ig protein solution compared to buffer alone (Figure 1−3). Our results are in agreement with other reports in the literature, where HPβCD has no effect on the aggregate formation in protein solutions or it is effective against aggregation induced by one type of stress while it has no effect on the other stresses.36,40,62 On the other hand, HPβCD shows a concentration based decrease in LLPS on DVD-Ig protein solution (Figure 5). In our previous study, it was observed that LLPS for DVD-Ig protein was mediated by both hydrophobic and dipolar interactions.49 At pH 6.6, protein carries a slight positive charge and this pH being close to the pI (∼7.5) would have a dipole moment associated with it, which is further confirmed by a low zeta potential (ζ = 2.8 mV) and strong attractive interactions from light scattering. These dipolar interactions decrease on increasing ionic strength of the solution (kD = −61.45 mL/g at 15 mM and kD = −38.12 mL/g at 50 mM ionic strength).49,63 Similarly, Tcloud (data not shown) decreased from 19.5 °C at 15 mM ionic strength to 14.25 °C at 50 mM ionic strength, indicating a decrease in dipole-mediated interactions in solution (35 mM sodium chloride was added to adjust the ionic strength to 50 mM). HPβCD is a neutral molecule and, therefore, should not affect the charges/dipole moment on the protein. On addition of 30 mM HPβCD, a decrease in Tcloud is more prominent (19.5 to 8.75 °C) than with the addition of salt/increasing ionic strength (19.5 to14.25 °C). Also, the decrease in Tcloud in the presence of increasing concentration of HPβCD in different buffers (constant ionic strength was maintained for histidine and phosphate buffer in this study) is similar. To further explore the mechanism, light scattering studies were also performed for DVD-Ig protein in the presence and absence of HPβCD as a function of temperature. Figure 7 shows a plot of kD measured at 11, 18, and 25 °C in buffer and 30 mM HPβCD. In buffer, protein solution exhibits strong attractive interactions in solution, which increases significantly as the temperature is lowered to 11 °C (kD = −135 mL/g). For a system exhibiting LLPS, as the temperature is lowered below the critical temperature (Tc), the coexistence curve becomes wider, and lower protein concentrations also tend to undergo phase separation. This is in agreement with increased attractive interactions between the protein molecules at low temper-

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DISCUSSION Effect of Excipients on Aggregation and LLPS. Mechanisms by which different excipients exert their stabilizing/destabilizing effects on protein aggregation have been thoroughly studied and reported in the literature.17,18 In this study, effect of different excipients (with reported different mechanism by which they affect aggregation) was investigated on aggregation and LLPS for DVD-Ig proteins solution. Preferentially Excluded Excipients. Both PEG and sucrose increase thermal stability of the protein in solution by preferential exclusion mechanism.19,28 In the presence of sucrose, though the overall amount of aggregates in DVD-Ig protein solution increased at 40 °C (Figure 3b), aggregation decreased compared to buffer alone, which is consistent with the literature reports.25,26 However, the opposite effect was observed for PEG, where both aggregation and fragmentation increased at 40 °C (Figure 3). PEG is a macromolecular crowding agent and exerts preferential exclusion by steric effect.20,27 Recent literature reports suggest that, in addition to steric effects (entropic effects), low molecular weight PEGs also show weak (nonspecific) binding to proteins (enthalpic effects, which may possibly lead to exposure of the fragmentation and/ or aggregation prone sites) thereby destabilizing it.51−53 Formation of peroxides and other chemical moieties (aldehydes and esters) in PEG solutions due to air exposure/aging and increased temperatures has also been reported in the literature and may promote covalent interactions in protein solutions, resulting in increased aggregation.54−56 Though both sucrose and PEG are preferential excluded cosolute, the mechanism by which sugar stabilizes the protein is different from PEG. Different rates of aggregation and fragmentation for PEG and sucrose at 20 and 40 °C suggest multiple aggregation mechanisms at play, which cannot be distinguished from the current data. From Figure 4, LLPS also increased in the presence of PEG (indicated by more negative kD values and larger Tcloud); the results are consistent with those reported in the literature, where PEG has been shown to shift the coexistence curve for proteins to higher temperatures.22,24 In contrast, sucrose reduced LLPS in solution as indicated by less negative kD and lower Tcloud values. Although sugars are polar molecules, they have some hydrophobicity associated with it, resulting in specific (weak) binding to aromatic amino acids and its derivatives (in comparison to aliphatic amino acids);57,58 therefore they reportedly have variable effects on solubility of proteins.58,59 Site specific binding of sucrose with the aromatic amino acids on protein surface may be responsible for reduced Tcloud and kD for DVD-Ig protein. Though we have described few possibilities of the opposing effects of PEG and sucrose on aggregation and LLPS, from the current data it is difficult to comment on the exact mechanism by which either of the excipients exerts its effect, and detailed 779

DOI: 10.1021/acs.molpharmaceut.5b00668 Mol. Pharmaceutics 2016, 13, 774−783

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aggregate formation increases with increasing temperatures while tendency to phase separate decreases. Earlier sections discussed the effect of solution conditions and excipients on aggregation and LLPS; this section discusses the relationship between aggregation and LLPS as a function of temperature. At 4 °C, all the samples exhibited liquid−liquid phase separation in the presence of excipients studied (Figures 4 and 5). On undergoing LLPS, two phases of different protein concentrations are formed and due to the Donnan effect excipients will also partition in two phases; concentration of the excipient in the upper phase or protein-poor phase will be much higher than in protein-rich phase. Ideally both high concentration of the protein and lower concentration of the excipient in protein-rich phase should accelerate the aggregation of proteins. However, no change in aggregate or fragment content was observed after the samples were incubated at 4 °C for 45 days (Figure 1). Though the concentrations of the two phases were different at 4 °C, no change in aggregate or fragment content was observed in the protein-rich phase analyzed after 45 days (data not shown). This indicates that LLPS does not result in aggregate formation at this temperature during this period (45 days stability studies). At 20 °C, increase in aggregate formation was observed for DVD-Ig protein solution in the presence of PEG 400, while at all other conditions monomer content remained the same after 45 days. From Figure 4, Tcloud for DVD-Ig protein in buffer and in the presence of Tween 80 is around 20 °C. Please note that, in the current study, Tcloud is a qualitative measure of the temperature that marks the onset of phase separation in solution at a constant concentration (16 mg/mL) and is the temperature where percent transmittance is 70. At 20 °C, though the solution exhibits significant opalescence at the two conditions (buffer alone and in the presence of Tween 80), phase separation may be in transient state due to kinetic hindrance (solution may be in the metastable region of the phase diagram approaching the stable region). On the other hand, Tcloud for PEG is around 25.5 °C and is distinctly phase separated at 20 °C. This indicates that though LLPS can accelerate aggregation in solution, temperature is the dominant factor in aggregation kinetics. This was further confirmed from SEC analysis of samples incubated at 40 °C, where significant increase in aggregate and fragment content was observed for DVD-Ig protein solutions at all conditions. However, all the samples were homogeneous and no liquid−liquid phase separation was observed at any of the conditions. At 20 °C, area under the curve (AUC) for PEG sample does not change from t0 to t45 (where aggregation was observed), further confirming presence of just soluble aggregates and no loss due to precipitation; while AUC changed at 40 °C, confirming formation of insoluble aggregates at higher temperature. Hence, at 4 °C, even though all the samples are phase separated, they do not aggregate as temperature affects the kinetic barrier for aggregate formation; while at 20 °C, both LLPS and increased temperature promote formation of irreversible, soluble aggregates in the solution. It is well-known that the rate of protein degradation pathways is accelerated at higher temperatures and forms the basis of accelerated stability studies. However, the prediction of degradation rates from higher temperatures may not be representative of the actual storage conditions, which is refrigerated condition for most proteins. Though many models are routinely used to extrapolate accelerated stability conditions

Figure 7. Plot of kD determined from dynamic light scattering as a function of temperature in the presence (dotted line) and absence (solid line) of HPβCD at pH 6.6 in histidine buffer with ionic strength of 15 mM. Protein concentration was varied from 0.1 to 10 mg/mL depending on the temperature so as to obtain linear plots of mutual diffusion coefficient (Dm) against concentration. 200 mM stock solution of HPβCD was prepared in histidine buffer and then diluted to maintain 30 mM concentration. All solutions were analyzed in duplicate, plot represents mean value, and error bars represent range. Error bars if not visible are smaller than the symbols used.

atures. At all three temperatures, HPβCD reduces attractive interactions in solution as indicated by less negative kD values. However, the effect is more prominent at 11 °C (ΔkD ∼ 46 units) than at 18 °C (ΔkD ∼ 22 units) and 25 °C (ΔkD ∼ 17 units), indicating reduced hydrophobic interactions at lower temperatures. Further lower temperatures could not be studied because, on decreasing the temperature, lower concentrations (∼6 mg/mL) also exhibit opalescence and a tendency to phase separate. This indicates that HPβCD interacts specifically with the protein, possibly with the hydrophobic groups on the protein surface, and exerts its effect independent of the buffer species. Literature reports suggest weak binding of HPβCD with aromatic amino acids on protein surface, thereby reducing attractive protein−protein interactions.64 From Tcloud measurements and DLS in the presence of HPβCD, concentration based decreases in attractive interactions were observed for DVD-Ig protein solutions, indicating specific binding of the excipient with the protein. These results are consistent with Tcloud and kD measured for DVD-Ig protein in the presence of sucrose, where decrease in attractive interactions in solution is due to specific binding to aromatic amino groups. Our studies indicate that the mechanism for LLPS and aggregation in protein solution is different and shows strong temperature dependence. Similarly, any excipient used in a formulation may interact with the protein by more than one mechanism depending on the protein itself (specific amino acids on protein surface) and other extrinsic factors, including but not limited to pH, ionic strength, protein concentration, excipient concentration, and temperature. Hence the selection of a suitable excipient for a formulation may not be straightforward and has to be investigated on a case by case basis. Effect of Temperature on Aggregation and LLPS. Liquid−Liquid phase separation of a solution into a proteinrich and protein-poor phase is more likely to promote protein self-association and/or irreversible aggregation since these phenomena are concentration dependent. However, LLPS and aggregation respond to the temperature in opposite ways; 780

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Molecular Pharmaceutics

(3) Vekilov, P. G. Phase transitions of folded proteins. Soft Matter 2010, 6 (21), 5254−5272. (4) Vekilov, P. G. Phase diagrams and kinetics of phase transitions in protein solutions. J. Phys.: Condens. Matter 2012, 24 (19), 193101. (5) Dumetz, A. C.; Chockla, A. M.; Kaler, E. W.; Lenhoff, A. M. Protein phase behavior in aqueous solutions: crystallization, liquidliquid phase separation, gels, and aggregates. Biophys. J. 2008, 94 (2), 570−83. (6) Roberts, C. J. Protein aggregation and its impact on product quality. Curr. Opin. Biotechnol. 2014, 30 (0), 211−217. (7) Jiskoot, W.; Randolph, T. W.; Volkin, D. B.; Middaugh, C. R.; Schöneich, C.; Winter, G.; Friess, W.; Crommelin, D. J. A.; Carpenter, J. F. Protein instability and immunogenicity: Roadblocks to clinical application of injectable protein delivery systems for sustained release. J. Pharm. Sci. 2012, 101 (3), 946−954. (8) Mahler, H. C.; Friess, W.; Grauschopf, U.; Kiese, S. Protein aggregation: pathways, induction factors and analysis. J. Pharm. Sci. 2009, 98 (9), 2909−34. (9) Li, Y.; Roberts, C. J. Protein Aggregation Pathways, Kinetics, and Thermodynamics. In Aggregation of Therapeutic Proteins; John Wiley & Sons, Inc.: 2010; pp 63−102. (10) Sanchez-Ruiz, J. M. Protein kinetic stability. Biophys. Chem. 2010, 148 (1−3), 1−15. (11) Narhi, L. O.; Schmit, J.; Bechtold-Peters, K.; Sharma, D. Classification of protein aggregates. J. Pharm. Sci. 2012, 101 (2), 493− 8. (12) den Engelsman, J.; Garidel, P.; Smulders, R.; Koll, H.; Smith, B.; Bassarab, S.; Seidl, A.; Hainzl, O.; Jiskoot, W. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm. Res. 2011, 28 (4), 920−33. (13) Sharma, V. K.; Kalonia, D. S. Experimental Detection and Characterization of Protein Aggregates. In Aggregation of Therapeutic Proteins; John Wiley & Sons, Inc.: 2010; pp 205−256. (14) Zölls, S.; Tantipolphan, R.; Wiggenhorn, M.; Winter, G.; Jiskoot, W.; Friess, W.; Hawe, A. Particles in therapeutic protein formulations, Part 1: Overview of analytical methods. J. Pharm. Sci. 2012, 101 (3), 914−935. (15) Weiss, W. F.; Young, T. M.; Roberts, C. J. Principles, approaches, and challenges for predicting protein aggregation rates and shelf life. J. Pharm. Sci. 2009, 98 (4), 1246−1277. (16) Wang, W.; Roberts, C. J. Aggregation of therapeutic proteins; Wiley Online Library: 2010. (17) Ohtake, S.; Kita, Y.; Arakawa, T. Interactions of formulation excipients with proteins in solution and in the dried state. Adv. Drug Delivery Rev. 2011, 63 (13), 1053−1073. (18) Kamerzell, T. J.; Esfandiary, R.; Joshi, S. B.; Middaugh, C. R.; Volkin, D. B. Protein−excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Adv. Drug Delivery Rev. 2011, 63 (13), 1118−1159. (19) Timasheff, S. N. Protein Hydration, Thermodynamic Binding, and Preferential Hydration. Biochemistry 2002, 41 (46), 13473−13482. (20) Bhat, R.; Timasheff, S. N. Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols. Protein Sci. 1992, 1 (9), 1133−1143. (21) Annunziata, O.; Asherie, N.; Lomakin, A.; Pande, J.; Ogun, O.; Benedek, G. B. Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (22), 14165−70. (22) Wang, Y.; Annunziata, O. Comparison between proteinpolyethylene glycol (PEG) interactions and the effect of PEG on protein-protein interactions using the liquid-liquid phase transition. J. Phys. Chem. B 2007, 111 (5), 1222−30. (23) Wang, Y.; Lomakin, A.; Latypov, R. F.; Laubach, J. P.; Hideshima, T.; Richardson, P. G.; Munshi, N. C.; Anderson, K. C.; Benedek, G. B. Phase transitions in human IgG solutions. J. Chem. Phys. 2013, 139 (12), 121904. (24) Wang, Y.; Latypov, R. F.; Lomakin, A.; Meyer, J. A.; Kerwin, B. A.; Vunnum, S.; Benedek, G. B. Quantitative evaluation of colloidal

to storage conditions and predict shelf life, several literature reports have cited the inefficiency of these models as reviewed in Roberts et al.65 In a recent paper, Saluja et al. have reemphasized the inadequacy of these models and have also highlighted the fact that these models do not take into consideration structure of multidomain proteins which do not follow simple two-step transition; hence, predicting aggregation rate for such molecules is even more challenging.66 Though we do not attempt to determine or correlate the aggregation kinetic behavior at different temperature conditions, the current study highlights the fact that at low temperatures possible liquid−liquid phase separation may alter the protein aggregation kinetics (especially for DVD-Ig protein as observed at 20 °C) over a long period of time. At 4 °C, aggregation kinetics are slow; hence sample analysis after 45 days may not be representative of long-term stability at this condition. For better prediction of protein shelf life either real time stability studies need to be performed or better predictive models, which take into account other physical instabilities at low temperatures, need to be developed.

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CONCLUSION HPβCD effectively lowers the phase separation temperature for DVD-Ig protein by selectively binding to aromatic groups on the protein surface, thereby decreasing the attractive protein− protein interactions between the molecules. However, HPβCD has no effect on temperature induced aggregation of proteins compared to that in buffer. Tween 80 has no effect on phase separation temperature, attractive interactions, or aggregate formation in solution. PEG 400 adversely affects both phase separation and aggregate formation in solution, while sucrose has positive effect and reduces both phase separation temperature for DVD-Ig protein and percent aggregates in solution. However, the mechanism by which these different excipients exert their effect (positive or negative) on aggregation and LLPS is different. Though no aggregate formation was observed at 4 °C, where solution exhibited phase separation, 20 °C data suggests that aggregate formation is accelerated on phase separation. Temperature plays a dominant role in both liquid−liquid phase separation and formation of soluble, irreversible aggregates in solution.

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AUTHOR INFORMATION

Corresponding Authors

*A.S.R.: phone, 347-331-8669; e-mail, ashlesha0105@gmail. com. *D.S.K.: phone, 860-486-3655; fax, 860-486-2076; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors would like to thank AbbVie Bioresearch Center, Worcester, MA, for material and financial support for the work.

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