Solubility Challenges in High Concentration Monoclonal Antibody

Sep 25, 2015 - *Phone: (650) 225-8912. Fax: (650) ..... Quartz windows were used; the scans were acquired at a wavelength of 230 nm (for 0.1 mg/mL sam...
4 downloads 9 Views 6MB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions Mariya Pindrus,†,∥ Steven J. Shire,† Robert F. Kelley,‡ Barthélemy Demeule,† Rita Wong,† Yiren Xu,§ and Sandeep Yadav*,† †

Late Stage Pharmaceutical Development, ‡Drug Delivery, and §Protein Analytical Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ∥ Summer Intern from Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, United States S Supporting Information *

ABSTRACT: The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue. KEYWORDS: solubility/precipitation, molecular interactions, interaction parameter (kD), mutant design, hydrophobic interactions, high concentration proteins

■

INTRODUCTION During development of biopharmaceuticals, high protein concentrations are very commonly encountered and pursued for various reasons. Such reasons include target indications requiring high dose, development of a subcutaneous (SC) product, and amenability for sustained delivery approaches to reduce injection frequency, thus gaining convenience for the patient. Other efforts are aimed to compound multiple drug product configurations from single drug substance bulk to mitigate storage constraints. Developing moderate to high concentration formulations brings forth several challenges due to solubility limitations, phase separation, increased viscosity, and aggregation propensity that may hamper the manufacturability, deliverability, stability, and shelf life of the product.1 Considerable progress has been made lately in understanding the viscosity,1,2 self-association,1,3,4 and aggregation propensity1,5−8 at high protein concentrations. However, addressing solubility limitations can be very challenging, especially when the formulation is intended for delivery into the SC space, where the intention is not only to keep the formulation stable over the shelf life but also to ensure the stability upon delivery into the SC © XXXX American Chemical Society

space until absorption. The solution behavior and stability of a protein molecule are primarily governed by the intermolecular protein−protein interactions (PPI), which in turn are dictated by the three-dimensional protein structure and the surrounding solution environment. The nature of PPI can range from longrange electrostatic interactions (which can be both attractive and repulsive in nature) to short-range hydrophobic interactions (attractive).9 Though hydrophobic forces are not strictly speaking an interaction, dehydration of a hydrophobic group is an entropically driven, energetically favorable process, effectively favoring a contact between hydrophobic surfaces.10−12 Strong protein−protein attractions can result in self-association and may decrease a protein’s solubility,13,14 thus limiting the concentration for administration as a therapeutic agent. Classically, solubility has been defined as a condition when the solution remains clear (transparent, uncolored) and does not gel or forms Received: May 1, 2015 Revised: August 17, 2015 Accepted: September 25, 2015

A

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics precipitate15 or crystals. Protein solubility in solution can depend on intrinsic factors,1 such as the nature of protein itself (i.e., its physical and chemical structure), as well as on numerous extrinsic factors1,16 such as solution pH, temperature, salt identity and concentration, and presence of excipients. It has been shown that protein solubility is governed by a balance of electrostatic and hydrophobic effect contributions.17 Furthermore, under different solution conditions, PPIs of various nature may be governing protein solution behavior. The need for higher protein concentrations to support SC formulations necessitates a comprehensive characterization and understanding of the intermolecular interactions affecting protein solubility before a molecule can become a successful drug candidate. The work presented in this article investigates the solubility challenges observed with one monoclonal antibody (mAb-G) and introduces the approach of utilizing biophysical measurements to understand the solubility behavior at high protein concentration. Expanding on the previous work that has correlated the interaction parameter (kD) to viscosity,2,18 an investigation was carried out using dynamic light scattering (DLS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) to understand the solution behavior, and demonstrate that characterizing interactions can serve as a qualitative predictive tool for assessing solubility issues at high concentration. Furthermore, another IgG1 (mAb-R) was examined that binds overlapping epitopes on the same target as mAb-G. The mAb-G was observed to have limited solubility at physiological pH and ionic strength, while the mAb-R did not show any turbidity or precipitation issues. To gain a better mechanistic understanding of the underlying cause leading to solubility issues, mutants of mAb-G were designed and tested. One complementarity-determining region (CDR) accounts for ∼67% of the CDR sequence difference between mAb-G and mAb-R. Amino acid substitutions or deletions within this single CDR were introduced to generate mutants and tested for modulating intermolecular protein−protein interactions and solubility behavior.

Methods. Mutant Design. Sequences of mAb-G and mAb-R were aligned to compare the differences between the two molecules. In view of the fact that the human IgG1 Fc framework of mAb-G is similar to other Genentech molecules, that do not precipitate at high ionic strength, the focus was on the differences in the complementarity determining regions (CDRs) of the molecules in an attempt to determine whether residues in the CDRs are responsible for precipitation of mAb-G in certain solution conditions. Differences in the CDRs of both heavy and light chain were noted between mAb-G and mAb-R, with 8 out of 12 amino acid differences (∼67%) observed in one CDR. The mutant GM-1 was created by replacing this CDR in mAb-G with the corresponding residues found in mAb-R (cartoon shown in Supplementary Figure S1A). This resulted in changes at eight positions, with overall decreased hydrophobicity of the CDR (hydrophobicity plots of mAb-G and mAb-R CDRs shown in Supplementary Figure S1B), but no net differences in charged residues or residues susceptible to deamidation or oxidation. Since a key change in GM-1 was replacement of two adjacent hydrophobic residues with small, polar residues, an additional mutant (GM-2) was constructed in which these two hydrophobic residues in mAb-G were deleted. For both mutants, residues responsible for UV absorbance (tyrosine, tryptophan, and phenylalanine) remained unchanged, justifying the use of the same absorptivity as that of mAb-G for determination of protein concentration. Sample Preparation. Samples were dialyzed into appropriate buffer conditions by performing at least four centrifugation cycles of buffer exchange using Amicon Ultracentrifugation tubes with MWCO of 30 kDa (EMD Millipore, Billerica, MA, USA). After dialysis was completed, the pH was verified to be within ±0.15 pH units from the targeted value. Samples were filtered through 0.22 μm Millex/Durapore filters (EMD Millipore, Billerica, MA, USA), and concentrations were determined at 280 nm using a SoloVPE (C Technologies, Inc., Bridgewater, NJ, USA) spectrophotometer. An absorptivity of 1.75 mL/(mg·cm) was used for mAb-G, GM-1, and GM-2 and 1.8 mL/(mg·cm) for mAb-R. Where necessary, the samples were diluted to appropriate concentrations required for each experiment. Solubility Studies. Solubility studies of mAb-G were performed in Pierce Slide-A-Lyzer dialysis cassettes (Thermo Scientific, USA) as a function of pH and ionic strength. For high ionic strength conditions, 150 mM NaCl was added to buffers, resulting in a total ionic strength of 165 mM. For high ionic strength solubility/precipitation experiments in glass vials and DLS experiments, the ionic strength was adjusted to ∼150 mM by spiking in NaCl (added ∼138 mM of NaCl, with 15 mM ionic strength contribution from the buffer resulting in the total ionic strength being ∼153 mM). The ∼153 mM solution ionic strength will be denoted as 150 mM ionic strength in the rest of the text for the ease of presentation, as at high ionic strength extra 3 mM is not expected to make a significant difference on solution properties and molecular behavior. All solubility studies were performed at 2−8 °C to reflect conditions typical for formulation storage. Dialysis Using Slide-A-Lyzer Dialysis Cassette. The mAb-G was dialyzed into low ionic strength (15 mM) buffers at pH 5, 6, 7, 8 (note: for pH 8 buffer preparations, both phosphate and tris were used) by loading 0.5 mL of mAb-G at a concentration of ∼91 mg/mL into 10k MWCO dialysis cassette and dialyzing overnight against 250 mL of respective buffers. The dialyzing buffer was replaced once with fresh buffer solution. For high ionic strength conditions, 150 mM NaCl was added to the

■

MATERIALS AND METHODS Materials. Monoclonal antibodies, mAb-G and mAb-R, and mutants, GM-1 and GM-2, were produced at Genentech and purified using a standard multistep purification process aimed at producing material of high purity (note that mAb-G is the same monoclonal antibody reported in previous studies18). Buffers were prepared at appropriate buffer strengths to maintain a solution ionic strength of 15 mM without addition of any salt. Sodium acetate and acetic acid were used for the pH 5 buffer, histidine base and histidine-HCl for pH 6, sodium phosphate, mono- and dibasic were used for pH 7 and pH 8, and tris base and tris-HCl were used for an alternative pH 8 buffer. The pH of the buffers, measured at room temperature, was verified to be within ±0.15 from the target value. Pierce Slide-A-Lyzer dialysis cassettes and Millipore Amicon Ultracentrifugation tubes were obtained from Thermo Scientific (Waltham, MA, USA) and MilliPore (Billerica, MA, USA), respectively. Sodium chloride (NaCl) was used to increase solution ionic strength, where necessary. All buffers were prepared using reagent quality materials and Milli-Q water (Elga PURELAB Ultra, Celle, Germany). Phosphate buffered saline (PBS) to mimic physiological solution conditions consisted of pH 7.2, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4. All buffers were filtered through 0.22 μm filters (Corning Inc., Corning, NY, USA; or equivalent). B

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

and B consisted of 0.1% TFA in water and 0.1% TFA in ACN, respectively. The 29 min runs performed using a binary gradient pump consisted of a 2 min isocratic hold at 36% mobile phase B, an 18 min linear gradient to 45% mobile phase B, followed by a 1 min column wash with 95% mobile phase B, and an 8 min equilibration at 36% mobile phase B. Signal was detected at 280 nm. Differential Scanning Calorimetry (DSC). A MicroCal (MicroCal LLC, Northampton, MA, USA) was used to obtain the thermal scans of the molecules. Proteins were diluted to 1 mg/mL at pH 5, 15 mM ionic strength. Thermal scans from 10 to 95 °C at the rate of 1 °C/min were performed, and change in the heat capacity was monitored as a function of temperature. The reference cell was filled with the buffer and accounted for buffer signal subtraction. Buffer baseline and instrumental effects were accounted for by loading buffer individually as both sample and reference, and subtracting the difference in two buffer wells from the sample measurement (previously adjusted by buffer). The results were analyzed using Origin software. Concentrations were measured for all protein samples, and profiles were normalized by concentration (in mg/mL). Since the MWs of mAb-G and GM-1 are similar (145.8 versus 145.3 kDa) the molar concentration was assumed to be directly proportional to mass concentration. Dynamic Light Scattering (DLS). A Malvern Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK) and Dynapro Reader Plus (Wyatt, Santa Barbara, CA, USA) were used for DLS measurements. Sample stock solutions and respective buffers were filtered through 0.22 μm filters and subsequently used to prepare concentrations ranging between 4 and 20 mg/mL. Samples were centrifuged at 10,000 rpm for 5−15 min before measurements. The Dynapro was used for mAb-R and some mAb-G DLS analysis, whereas the rest of the DLS analysis was carried out using the Zetasizer. Dynapro utilizes an 830 nm laser as a light source with 158° backscatter signal acquisition. For measurements with Dynapro, 60 μL of each solution were loaded into individual wells of a sterile, 384-well, glass-bottom Greiner plate (Greiner bio-one, Monroe, NC, USA). An average of three readings was taken with an acquisition time of 20−30 s per measurement. The entire plate was read again at least one more time, to obtain replicate readings. The Malvern Zetasizer uses a 633 nm He−Ne laser with 173° backscattering data acquisition avalanche photodiode detector.20 For measurements with the Zetasizer, 75 μL of each solution were loaded into microquartz cuvette DTS2145 (Malvern Instruments, UK). Measurements were performed in automatic mode for a total of five replicates for each sample, with an average of 12−15 acquisitions for an individual sample. The DLS analysis was performed at 25 °C on both instruments. The Malvern DTS and Wyatt’s Dynamics software were used to analyze the Zetasizer and Dynapro data, respectively. In both cases the autocorrelograms were acquired and used to obtain the mutual diffusion coefficient (Dm). At relatively low protein concentrations, Dm can be related to the sample mass concentration, c (g/mL), the interaction parameter, kD (mL/g), and the self-diffusion coefficient, Ds (diffusion of a single molecule, in the limit of infinite dilution, measured in μm2/sec or equivalent), as follows:21

dialyzing buffer (total 165 mM), and the dialysis was performed overnight. Additionally, in order to compare the solubility behaviors of mAb-G, mAb-R, and designed mutants GM-1 and GM-2 in simulated physiologically conditions, 0.5−0.6 mL of protein solutions, at ∼89−100 mg/mL were extensively dialyzed against PBS, pH 7.2 for up to 72 h. In all cases, cassette dialysis was carried out at 2−8 °C, with a constant gentle stirring of the dialyzing buffer. Images of the cassettes were taken at room temperature, immediately after removal from the cold room. Solubility in Vials. Solutions of mAb-G at low ionic strength (15 mM) at pH 5, 6, 7 and 8 (phosphate and tris) were spiked with NaCl at room temperature as previously described. The spiked mAb-G solutions, at concentrations between 67 and 81 mg/mL (actual concentrations listed in Figure 3), were placed at 2−8 °C overnight. The images were taken at 2−8 °C. Turbidity Evaluation. Turbidity of solutions was evaluated by visual examination only. All descriptions of solutions as turbid or clear are based on visual evaluation, as no quantitative measurements were performed. Size Exclusion Chromatography (SEC). SEC analysis was carried out on an Agilent 1200 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), using a Tosoh Haas TSK G3000 SWXL column (Tosoh Biosciences LLC, King of Prussia, PA, USA), with dimensions of 7.8 mm × 30 cm. An isocratic method was used, with a 200 mM potassium phosphate, 250 mM potassium chloride, and pH 6.2 ± 0.1 mobile phase, at a flow rate of 0.3 mL/min. Samples were diluted to 2 mg/mL in the mobile phase, and 25 μL were injected. The sample compartment was kept at 2−8 °C, and separation analysis was performed at room temperature using UV absorption detection at 280 nm. Chromeleon software (Thermo Scientific, Sunnyvale, CA, USA) was used for analysis and quantitation of the chromatograph to determine the purity of the material (as % monomer peak). Ion Exchange Chromatography (IEC). IEC analysis was carried out using an Agilent 1200 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), using a Dionex ProPac WCX-10 column (Thermo Scientific, Waltham, MA, USA), with 4 mm × 25 cm dimensions. Samples were diluted to 1 mg/mL and treated with carboxypeptidase prior to loading 50 μL injection volume onto the column. Two mobile phases were used: mobile phase A consisting of 25 mM potassium phosphate, pH 6.9 ± 0.05, and mobile phase B consisting of 25 mM potassium phosphate and 120 mM potassium chloride at pH 6.9 ± 0.05. A binary pump was utilized to start an elution gradient at 90% A, 10% B mobile phase composition (condition at which sample was injected onto a column), followed by a linear gradient to 50% B over 40 min, and then followed by an instant (0.1 min) switch to 100% B, which was held for the next 5 min. The buffer composition was then switched back to the initial condition to re-equilibrate the column for 10 min prior to the next injection. The autosampler temperature was set to 2−8 °C, and the column compartment was kept at 40 °C. The signal was detected at an absorbance of 280 nm. The chromatography was performed at a constant flow rate of 0.5 mL/min, and Chromeleon software was used for analysis of the chromatogram. Reversed Phase HPLC (RP-HPLC). RP-HPLC analysis was performed on an Agilent 1200 HPLC system (Palo Alto, CA, USA) as previously described.19 Briefly, a Pursuit 3 diphenyl reversed phase column with 150 × 4.6 mm, 3 μm dimensions (Varian, Lake Forest, CA, USA) was placed into a temperaturecontrolled column compartment set to 75 °C. Mobile phases A

Dm = Ds(1 + kDc) where the kD and Ds can be obtained from the slope and/or intercept of a Dm versus c plot.18 The value of kD reveals the nature of the interactions, with a positive kD signifying C

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

S20,w versus concentration plot yields a value for S°20,w, which is the sedimentation coefficient in the limit of infinite dilution, thus representative of size of a single molecule, unaffected by intermolecular interactions. Linear regression analysis was performed using Kaleidagraph (Synergy software, Reading, PA, USA) with the general curve fit mode to obtain errors in the parameters of the fit. Molecular Charge Determination. The electrophoretic mobilities of the mAbs were determined at pH 5, 6, 7, and 8 at 15 mM solution ionic strength using a Mobius mobility instrument (Wyatt, Santa Barbara, CA, USA). The measurements were performed at 0.5 mg/mL mAb concentration at room temperature. The electrophoretic mobility was used to calculate the effective charge on the molecule (Z*e, or ZDHH) using the Debye−Hückel Henry relationship18

intermolecular repulsions and negative kD representing attractive interactions. While the kD parameter accounts for some contribution from protein solute and protein solvent interactions, to a good approximation, kD can be assumed to represent the nature of the PPI. Although hydrodynamic drag also contributes to a negative kD value, for mAbs a kD magnitude more negative than −5.34 mL/g depicts contribution of attractive interactions to the interaction parameter.18 The protein concentrations of all dilutions were measured for kD calculations. The pH of 150 mM sample preparations (and some of 15 mM) was measured to discern whether pH shifts occurred upon spiking with NaCl (especially for phosphate buffers). For minor pH shifts, the pH of highest and lowest concentration sample was averaged and reported. Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC). SV-AUC was utilized to evaluate overall size as well as association level of the proteins of interest. A Beckman Coulter Proteome Lab XL-I Analytical Ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) was used to assess hydrodynamic behavior of the molecules using absorbance optics. Samples were diluted to a range of concentrations between 0.1 and 0.5 mg/mL in SEC mobile phase. Experiments were performed in centrifuge cells equipped with 12 mm graphite-filled Epon centerpieces (Spin Analytical, Durham, NH, USA) at 20 °C using a rotor speed of 30,000 rpm. Quartz windows were used; the scans were acquired at a wavelength of 230 nm (for 0.1 mg/mL samples) and 280 nm (for all other sample concentrations) at 30 μm radial increments. The sedimentation boundaries were analyzed with SEDFIT (version 11.3 and 11.72c, http://www. analyticalultracentrifugation.com). The resulting continuous c(s) distribution with 90% confidence level was calculated after optimizing baseline, meniscus, and cell bottom positions by nonlinear regression. Although SEDFIT is used for a discrete species model, if the kinetics of the association is slower than the rate of sedimentation, this model may be an acceptable estimation. SEDFIT has been previously used for antibody reversible interaction systems as recently documented.22 All s values obtained with the c(s) distribution were converted to S20,w with SEDNTERP (http://sednterp.unh.edu) using the measured density (ρ) and viscosity (η) of buffer and protein partial specific volume (ν̅ = 0.73 mL/g) estimated from amino acid composition as follows:23,24

Z*e = 6πηrhμE

1 + κrh f1 (κrh)

where η is the solution viscosity, rh is the hydrodynamic radius, κ is the inverse Debye length, μE is the electrophoretic mobility, and f1(κrh) is Henry’s function. Dynamics software was used to analyze the data. The calculated charge (ZDHH) was compared between mAb-G and mAb-R, as well as to each molecule’s respective theoretically calculated charge value. The theoretical charge was computed assuming independent titrations of the individual ionizing amino acids and intrinsic pKa values based on model compound hydrogen ion titration data.25

■

RESULTS Solubility Behavior of mAb-G and mAb-R at High Concentration: Effect of Solution pH and Ionic Strength. To assess solution behavior of mAb-G under relevant physiological pH and ionic strength conditions, mAb-G at 89 mg/mL was dialyzed into PBS (pH 7.2, ∼150 mM ionic strength) for 24 h, which resulted in extensive turbidity of the mAb-G solution (Figure 1A). To elucidate whether the increase in turbidity upon exposure to PBS was reversible or due to an irreversible phase transition, the same dialysis cassette (shown in Figure 1A) was buffer exchanged into pH 5, 15 mM ionic strength buffer. Upon dialysis into pH 5 the mAb-G solution clarified (Figure 1B). Although Figure 1B shows this result after 15 h of dialysis, the solution actually clarified after only 2 h of transfer to the pH 5 dialysis buffer, demonstrating the reversible nature of the phase transition under these conditions. The mAb-R solutions, however, remained clear when concentrated (∼90 mg/mL) and dialyzed against PBS (Figure 1C). To dissect whether the observed solution behavior of mAb-G in PBS could be attributed to the pH or the ionic strength of

⎛ η ⎞ (1 − νρ) ̅ 20,w T,B ⎟ S20,w = ST,B⎜⎜ ⎟ (1 − νρ) η ̅ T,B ⎝ 20,w ⎠

where T in the subscript stands for experimental temperature, B for buffer, w for water, and 20,w for standard conditions of water at 20 °C. The extrapolation to zero concentration of the

Figure 1. (A) mAb-G dialyzed in phosphate buffered saline (PBS) for 24 h; (B) same dialysis cassette as in panel A dialyzed in pH 5.0, 15 mM ionic strength buffer for 15 h (note: although image was taken after 15 h of dialysis, the solution became clear after only 2 h); (C) mAb-R dialyzed in PBS for 48 h. The hatched and crossed lines in panels B and C are due to membrane being stretched during dialysis. D

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 2. mAb-G concentrated solution (∼91 mg/mL) dialyzed at different solution pH at (A) 15 mM solution ionic strength and (B) 165 mM solution ionic strength adjusted with NaCl. *pH 8 (P), phosphate buffer; pH 8 (T), tris buffer.

Figure 3. mAb-G concentrated solutions in vials adjusted to 150 mM solution ionic strength with NaCl at different solution pHs. The concentration at different solution pHs was as follows: pH 5−80 mg/mL; pH 6−75 mg/mL; pH 7−81 mg/mL; pH 8 (P)−72 mg/mL and pH 8 (T)−67 mg/mL. *pH 8 (P), phosphate buffer; pH 8 (T), tris buffer. Note: vials containing buffer and pH 5 solution are upright, vials with pH 6−8 solutions are inverted.

the kD values for mAb-R changed from positive to negative (Table 1 and Figure 4A). Relatively lower kD at higher pH suggests more attractive PPI. Addition of salt to the mAb-R solution resulted in a change from repulsive to attractive interactions at pH 5 and pH 6, whereas other pHs showed a marginal decrease in intermolecular attractions (Figure 4B). The weakness of attractive interactions for mAb-R molecules at high ionic strengths possibly allows solutions to remain clear when concentrated and dialyzed against PBS (Figure 1C). At low ionic strength, mAb-G behaved similarly to mAb-R, exhibiting repulsive interactions at pH 5, which progressively became more attractive as the pH was increased to pH 8 (Table 1 and Figures 4A,B). Although the pH dependency of the interactions was similar between mAb-G and mAb-R at low ionic strength, the magnitude of interactions was stronger for mAb-G relative to that of mAb-R at all pH conditions. Furthermore, mAb-G displayed different behavior from that of mAb-R under high ionic strength conditions (Table 1 and Figure 4B). Both mAb-G and mAb-R at solution pH 5 and pH 6 showed a transition to more attractive interactions with increase in ionic strength; however, the magnitude of the attractions at pH 6 was relatively higher in mAb-G compared to mAb-R. Interestingly, for mAb-G, addition of 150 mM NaCl at pHs higher than 6 led to an increase in intermolecular attractions, unlike mAb-R. It is noteworthy to indicate that all the solutions where solubility of mAb-G was compromised showed a kD magnitude of 98% (Supplementary Figure S2). However, GM-1 showed a slightly longer retention time than mAb-G. This may be due to somewhat different interaction with the SEC column for the two molecules or could result from some conformational difference (smaller hydrodynamic size) of GM-1 compared to mAb-G. Additional studies using SV-AUC and DSC were performed to understand this aspect. Figure 5 shows the sedimentation coefficients (S20,w) of mAb-G and GM-1 at several concentrations prepared by dilution in SEC mobile phase (pH 6.2, high ionic strength). The mAb-G showed a higher sedimentation coefficient at all concentrations than GM-1, which is indicative of self-associating behavior. However, to estimate the effective size devoid of any solution nonidealities the S20,w was extrapolated back to zero concentration (S°20,w) using a linear regression analysis. The goodness of the fit is reflected by the random distribution of the residuals (inset of Figure 5) and low error calculated for the fitted parameters. The determined S°20,w values indicate that both molecules have essentially the same hydrodynamic size (mAb-G S°20,w = 6.3 ± 0.04, and GM-1 S°20,w = 6.24 ± 0.01). Further, the overall thermal scan profile (Supplementary Figure S3) was comparable between mAb-G and GM-1, with essentially the same melting temperature (Tm) for both molecules at pH 5, also suggesting that both molecules were equally compact. These results suggest that the differences in retention time for mAb-G and GM-1 are possibly due to minor differences in interaction with the SEC column matrix.

Figure 4. Interaction parameter, kD, for mAb-G and mAb-R as a function of buffer pH (pH 5 to pH 8) at ionic strength of (A) 15 mM and (B) 150 mM. Bars in black represent mAb-G data, while striped bars are of mAb-R data. For mAb-R at pH 8 only phosphate was used. Note: pH values were measured for 150 mM samples (as the average of highest/lowest concentration samples, described in Methods) and were as follows: mAb-G, 5.00, 6.16, 6.75, 7.55(p), 8.13(t); mAb-R, 4.97, 6.12, 6.70, 7.51(p). pH8(p), phosphate buffer; pH8(t), tris buffer.

difference between theoretical and experimentally measured charges for the mAbs is outside the scope of this work. These differences could be ascribed to various aspects, such as ion binding, electrostatic effects resulting in pKa shift of the ionizable residues, charge regulation, etc., and will be presented separately. Mutant Characterization. To ensure the purity and identity of the generated mutant GM-1, the mutant was characterized by F

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 5. Sedimentation coefficients of mAb-G and GM-1 as a function of protein concentration (c) from SV-AUC evaluation. Linear regression analysis and residuals to the fit are shown in inset.

bonds in mAb-G may influence the solubility of the molecule, possibly due to a local change in structure or dynamics of the molecule. To address whether the incomplete disulfide bond formation observed in mAb-G is not a major factor for the observed limited solubility, a study was carried out, setup and results of which are diagrammed in Figure 8. The mAb-G was concentrated to 97 mg/mL and subsequently dialyzed against PBS. As observed earlier, the solution became turbid. After centrifugation at 3000 relative centrifugal force a clear supernatant phase and turbid “precipitate” phase could be easily distinguished. A sample of the clear supernatant fraction was withdrawn, and free thiol content was determined by the RP-HPLC thiol assay. Supernatant was further concentrated to 70 mg/mL and again dialyzed against PBS to investigate whether precipitation would occur at the higher concentration. If the precipitation behavior was influenced by the free thiol content, the precipitate fraction would be enriched with molecules containing higher free thiols, while the supernatant would be expected to contain molecules with higher levels of fully formed disulfide bonds. It then follows that the supernatant, enriched with molecules having intact disulfide bonds, would not become turbid or precipitate when concentrated and dialyzed into PBS. It was, however, observed that the free thiol content of the mAb-G supernatant was identical to that of unmanipulated mAb-G. Also, upon concentration and further dialysis into PBS, the supernatant became turbid. Identical disulfide content in clear supernatant as that of unmanipulated mAb-G, as well as repeated precipitation observed upon further concentration of supernatant, served as proof that the disulfide bond content does not govern solubility behavior of mAb-G. Intermolecular Interactions for Mutants GM-1 and GM2 by DLS. The kD values determined for mAb-G, mAb-R, GM1, and GM-2 at different solution conditions are tabulated in

The IEC chromatograms show that the entire profile of GM-1 was shifted significantly to the more basic (right) side on the IEC chromatograph (Figure 6). It was also noticed that the overall basic content was higher, and acidic content was lower, several acidic variants were missing (in addition to peaks which were not as well-defined) in the GM-1 mutant relative to mAb-G. RP-HPLC shows (Figure 7) very different chromatograms between mAb-G and GM-1. The profile of another mutant, GM-2, was similar to that of GM-1 (Figure 7). Essentially one peak was present for GM-1 and GM-2, whereas mAb-G showed three peaks at 68%, 28%, and 4% of total area. This three-peak profile is typical for that previously reported for mAb-G19 and reflects the presence of an unformed disulfide bond between cysteines 22 and 96 in the VH domain. Although the presence of free thiols does not have an effect on binding or potency of the antibody,19 the negative charge from the ionized thiols would present as a more acidic antibody by IEC. The more basic IEC elution position of GM-1 is consistent with a fully formed C22−C96 disulfide bond in this molecule. In summary, the SEC, AUC, and DSC data indicated that the designed GM-1 mutant was sufficiently pure and without any gross conformational changes. The minor differences in IEC can be explained by the change in total free thiol content of the designed mutants as indicated by the RP-HPLC analysis.19 A study of the free thiol content in mAb-G and how it relates to the observed solubility issue is described below. Further investigations as to how these mutations result in a fully formed C22−C96 disulfide bond in GM-1 and GM-2 are in progress. Impact of Total Free Thiol Content on Solubility Behavior of mAb-G. The presence of free thiol content in the poorly soluble mAb-G suggests that the unformed disulfide G

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 6. (A) Ion exchange chromatography (IEC) profiles of mAb-G and GM-1. Zoomed in portion is shown in inset. (B) Peak distribution of various species as % area of entire peak.

was more negative than ∼−20 mL/g, it was anticipated that concentrated mutants should remain clear in higher ionic strength conditions. Solubility Behavior of the GM-1 and GM-2 at High Concentration: Correlation with the Interaction Parameter (kD) and Specific Amino Acid Residues. The GM-1 solution at 89 mg/mL remained clear at 2−8 °C as well as 25 °C, i.e., no turbidity or gel-formation was observed after 24.5 h (Figure 9A) and 46 h (Figure 9B) of dialysis into PBS at 2−8 °C followed by warming to room temperature. Similarly, GM-2 at ∼100 mg/mL was dialyzed into PBS at 2−8 °C for 72 h and did not show any turbidity or gel formation both under refrigerated condition and at room temperature (Figure 9C). The mAb-G solution at 89 mg/mL after a 24 h dialysis into PBS at 2−8 °C (Figure 9D) is included in the figure to demonstrate the difference in solubility between mAb-G, GM-1, and GM-2. The observed solubility behavior of GM-1 and GM-2 at high protein concentration and ionic strength compares well with

Table 1. The interaction parameter (kD) determined from DLS at 15 mM buffer ionic strength revealed that at all pH conditions tested, intermolecular interactions, and self-associating behavior of the mutant GM-1 resembled mAb-R more than they did mAb-G. Notably, at pH 7 both GM-1 and GM-2 mutants had similar kD values to mAb-R. DLS analysis at 150 mM ionic strength was carried out to examine the effect of high solution ionic strength on intermolecular interactions (Table 1). At pH 5 and 6, high ionic strength modulated GM-1 intermolecular attractions very similarly to what was observed with mAb-R. At pH 7, contrary to mAb-R, GM-1 and GM-2 showed an increase in intermolecular attractions at high ionic strength; the magnitude of the effect, however, was smaller than what was observed for mAb-G and the interaction parameters were less negative. Considering that kD values for GM-1 and GM-2 were less negative than −10 mL/g under all conditions investigated, while solubility issues were only observed in cases where kD H

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 7. Disulfide content evaluation of mAb-G, GM-1, and GM-2 by reverse phase HPLC (RP-HPLC). Arrows designate peaks representative of various degree of disulfide bond formation.

Figure 8. Evaluation of the impact of free thiol content (unformed disulfide bonds) on solubility of mAb-G: schematic of experimental design and results postdialysis in phosphate buffer saline.

Figure 9. Solution behavior of mAbs dialyzed into PBS solution at high protein concentration for (A) GM-1 mutant after dialysis for 24.5 h; (B) same dialysis cassette of GM-1 mutant after dialysis for 46 h; (C) GM-2 after dialysis for 72 h; (D) mAb-G after 24 h of dialysis. Note: 24 h time point was used as a minimum time for assessment. Longer times were investigated to corroborate behavior (e.g., solutions remaining clear even for longer times).

mediated thermodynamic contribution to solution nonideality. An increase in attractive intermolecular interactions revealed by a negative B22 has been shown to correlate with a decrease in solubility.13,14,26 Analogous to B22 is the nonideality interaction parameter kD, which constitutes both thermodynamic and hydrodynamic contributions to solution nonideality. Similar to B22, the sign and magnitude of kD reveals the nature and the strength of the molecular interactions in a solution and can be determined using DLS measurements. DLS is a dilute solution

weak intermolecular attractions revealed by the interaction parameter (kD) measured by DLS.

■

DISCUSSION

Molecular Interactions. Solubility and its relationship to intermolecular interactions has been studied and correlated previously with the second virial coefficient (often referred to as B, B2, or B22).13,14,26 B22 constitutes the chemical potential I

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

ionic strength. The involvement of strong intermolecular attractions resulting in limited solubility of mAb-G is further corroborated by the behavior of designed mutants. Substituting one of mAb-G’s CDRs (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2) resulted in molecules that did not show strong intermolecular attractions at high ionic strengths and resolved the solubility issue associated with mAb-G at high concentrations. Role of Hydrophobic Interactions on Solubility of mAb-G, mAb-R, and Mutants GM-1 and GM-2. No major differences were observed in the electrostatic surfaces of mAb-G and mAb-R using homology models (homology modeling method and models given in Supplementary Figure S4). Theoretical prediction of similar electrostatic surfaces was confirmed by experimentally measuring the effective charge associated with mAb-G and mAb-R at various pH conditions. The calculated and measured charge, as well as electrostatic surfaces showed that there were no major differences between the charges associated with mAb-G and mAb-R at all pH values, specifically at pH 7. Based on these assessments, it appears that at high ionic strengths the electrostatic interactions were not the driving force for strong attractions and precipitation behavior of mAb-G. A potential explanation for atypical behavior of mAb-G is that hydrophobic interactions make a dominant contribution to self-association when repulsive electrostatic interactions are screened at higher ionic strength. A sequence alignment (data not shown) shows that out of six CDRs total, two of the CDRs of mAb-R and mAb-G are identical in amino acid sequence, a third has a single conservative change, and the remaining three CDRs are more hydrophobic (Kyte−Doolittle scale32) in mAb-G. The involvement of hydrophobic residues in the poor solubility of mAb-G is further corroborated by the solution behavior of the designed mutants. A mutant molecule GM-1 was created whereby one of the CDR loops of mAb-R was substituted into mAb-G. Solubility experiments confirmed a lack of precipitation for GM-1. Thus, solubility differences between mAb-G and mAb-R appear to be related to sequence differences in one CDR. As indicated earlier, a key change in GM-1 was the replacement of two adjacent hydrophobic positions with small polar residues. An additional mutant (GM-2) was constructed in which these two hydrophobic residues in mAb-G were deleted. Interestingly, interactions and solubility behavior of the GM-2 mutant at pH 7 were very similar to those of the GM-1 mutant, both at low and high ionic strength conditions (Table 1). This observation suggests that deletion of two hydrophobic residues may be sufficient to reduce hydrophobicity of mAb-G and to prevent strong attractions at high solution ionic strength. Such an impact on solubility caused by very slight changes in sequence is not unusual as shown by the examples of solubility of hemoglobin A (HbA) versus S (HbS)33 as well as others.34,35 Although not investigated in this work, it is likely that as a result of such specific localized interactions the protein conformation of protein aggregates and precipitates may retain much of their native-like structure, to allow for reversibility once the solution conditions are changed to disfavor self-association and precipitation. As predicted, turbidity of mAb-G was observed to be reversible upon dialysis into low ionic strength solution at pH 5 (Figure 1B). Among other factors, protein solubility is a result of the interplay of two antagonistic effects: hydrophobicity and polarity.17 Proteins are folded in a manner as to hide hydrophobic

technique, which requires small sample volumes and thus small amounts of protein for analysis, yet the utility of DLS extends beyond low protein concentration solutions. For example, kD has been successfully used to scan different mAbs under various solution conditions for intermolecular interactions and relate PPI to solution viscosity at high protein concentrations, wherein large negative kD was indicative of a viscous solution, while positive or slightly negative kD resulted in much lower solution viscosity.2,18,27 Herein we have shown that kD is also useful for understanding solubility behavior, both in assessing antibodies for precipitation at high concentration and in understanding the nature of the underlying molecular interactions. Furthermore, these findings could warrant the use of DLS for assessing solubilities at high concentrations especially during early development when protein quantities are limited. The experimentally determined pI of mAb-R is 9.2. At pH 5, 15 mM ionic strength and away from its pI, a net positive charge associated with mAb-R resulted in the presence of net intermolecular repulsions (positive kD values). As the pH increased from pH 5 to 8, the net charge decreased and the interactions gradually became net attractive. Increasing the ionic strength to 150 mM screened the net charge associated with the molecules, either by Debye shielding or ion binding, resulting in a decrease in electrostatic interactions, which is in line with previous observations.28−31 The experimentally determined pI of mAb-G is 9.0. The solution behavior of mAb-G was similar to mAb-R at low ionic strength, i.e., intermolecular interactions transition from repulsive to attractive interactions as the solution pH increases from pH 5 to pH 8. However, the net attractions were significantly stronger for mAb-G (e.g., kD = −15.18 mL/g at pH 7) compared to mAb-R (e.g., kD = −7.75 mL/g at pH 7). The behavior under high salt conditions was also different for mAb-G compared to that observed for mAb-R (except for pH 5 conditions). The net attractive interactions induced by salt addition at pH 6 were much stronger for mAb-G (kD = −21.16 mL/g) compared to mAb-R (kD = −1.12 mL/g). At both pH 7 and 8, the addition of salt to mAb-G led to even stronger intermolecular attractions. This is potentially driven by the decrease of electrostatic repulsion due to counterion screening at higher ionic strengths, thereby reducing the effect of negative energy of approach and allowing molecular surfaces to come into closer contact. At shorter distances, entropically driven dehydration energy of hydrophobic regions can contribute significantly toward a favorable interaction between hydrophobic patches, effectively resulting in a net attraction between molecules.11,12 Since the CDRs as well as overall surface hydrophobicity of mAb-R is lower than mAb-G, the intermolecular attractions of mAb-R did not show an increase with increasing ionic strength. Solution Behavior at High Protein Concentration. Generally, lower kD values (representative of stronger intermolecular attractions) were associated with observations of insolubility. For example, relatively weak attractions of mAb-G at pH 5, 150 mM ionic strength (Table 1 and Figure 4B) resulted in a clear solution when dialyzed to 165 mM conditions at high protein concentration (Figure 2B). However, strong attractions observed for mAb-G at high ionic strength conditions at pH 6 and above (Table 1 and Figure 4B) resulted in precipitation and gel formation when concentrated solutions were dialyzed into 165 mM ionic strength buffers (Figure 2B). The mAb-R solutions, which showed relative weak intermolecular attractions at high ionic strengths, did not result in turbidity or precipitation when concentrated at high solution J

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Finally, it seems that substitution of one CDR was sufficient to mitigate solubility issues associated with mAb-G, and the rest of the CDRs need not be modified. It was further shown that simply removing two hydrophobic residues in one CDR of mAb-G was sufficient to prevent precipitation and demonstrated how minor sequence changes can have a huge impact on properties of a protein.

regions in the interior of the protein and surface-expose charged portions of the sequence.36 However, molecules are dynamic structures, and as they undergo normal molecular motions, some hydrophobic patches may become solvent exposed.36 In order to minimize the free energy of the system, the protein will need to refold or interact with other molecules, effectively hiding hydrophobic patches away from the surface.36,37 Such hydrophobic interactions, if present to a significant extent, can result in formation of higher order species,36 and eventually may reduce the solubility of the protein. In addition to the influence of intrinsic factors, protein−protein interactions and solubility can be affected by extrinsic factors as well. Considering the interplay of antagonistic forces of hydrophobicity and polarity, extrinsic factors, such as salts, can play a dual role on protein solubility. At low salt concentration (typically below ∼50 mM), addition of salt can increase solubility of the protein (the socalled salting-in region).16 At higher salt concentration the counterions tend to completely screen charges present on protein molecules, thereby minimizing charge repulsions. This allows molecules to come closer together, and entropy driven dehydration may dominate the energetics of molecular interactions by favoring interaction between hydrophobic patches, causing higher-order species formation and thus decreasing solubility (so-called salting out region16,17). As both hydrophobicity and polarity play a role, the salt concentration at which solubility decreases will be affected by the interplay of those forces. If at a given solution condition hydrophobic interactions are much stronger than electrostatic, the solubility limit will be reached even at low salt concentrations.17 Decreases in solubility could be due to how the interplay of intrinsic properties (e.g., protein structure) and extrinsic properties (e.g., solution conditions) affects molecular interactions and selfassociations, and the outcome is unique to each protein. Effect of Sequence Modification on Activity. The changes in the CDRs resulted in reduced biological activity for GM-1 and GM-2 (data not shown); it is yet to be determined if changes in other amino acids can increase activity while maintaining solubility achieved by deletion of responsible hydrophobic residues.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00336. Homology modeling and electrostatic potential calculations, modeling of electrostatic surfaces, and Figures S1−S4 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (650) 225-8912. Fax: (650) 742-1504. E-mail: yadav. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Authors wish to thank Thomas Patapoff, Brian Connolly, Karen Vigeant, Fred Lim, Zephania Kwong, Devendra S. Kalonia and Chris Williams for insightful discussions and help with some experimental aspects, Anthony Tomlinson for help with the figures, and Genentech Inc. for funding the internship project.

■

REFERENCES

(1) Shire, S. J.; Shahrokh, Z.; Liu, J. Challenges in the development of high protein concentration formulations. J. Pharm. Sci. 2004, 93, 1390−1402. (2) Yadav, S.; Shire, S. J.; Kalonia, D. S. Viscosity behavior of highconcentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects. J. Pharm. Sci. 2012, 101, 998−1011. (3) Liu, J.; Nguyen, M. D.; Andya, J. D.; Shire, S. J. Reversible selfassociation increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J. Pharm. Sci. 2005, 94, 1928−1940. (4) Kanai, S.; Liu, J.; Patapoff, T. W.; Shire, S. J. Reversible selfassociation of a concentrated monoclonal antibody solution mediated by Fab−Fab interaction that impacts solution viscosity. J. Pharm. Sci. 2008, 97, 4219−4227. (5) Wang, W. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm. 2005, 289, 1−30. (6) Minton, A. P. Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations. J. Pharm. Sci. 2005, 94, 1668−1675. (7) Saluja, A.; Badkar, A. V.; Zeng, D. L.; Kalonia, D. S. Ultrasonic rheology of a monoclonal antibody (IgG2) solution: Implications for physical stability of proteins in high concentration formulations. J. Pharm. Sci. 2007, 96, 3181−3195. (8) Saluja, A.; Sadineni, V.; Mungikar, A.; Nashine, V.; Kroetsch, A.; Dahlheim, C.; Rao, V. M. Significance of Unfolding Thermodynamics for Predicting Aggregation Kinetics: A Case Study on High Concentration Solutions of a Multi-Domain Protein. Pharm. Res. 2014, 31, 1575−1587. (9) Chari, R.; Jerath, K.; Badkar, A. V.; Kalonia, D. S. Long-and shortrange electrostatic interactions affect the rheology of highly concentrated antibody solutions. Pharm. Res. 2009, 26, 2607−2618. (10) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; J. Wiley: New York, 1980.

■

SUMMARY AND CONCLUSIONS This work evaluated the solubility issues associated with an IgG1 (mAb-G) at high solution ionic strengths and high protein concentration and established the relationship between intermolecular interactions and amino acid sequence in the CDRs. The importance of amino acid sequence was highlighted by comparing the CDRs of mAb-G with the CDRs of another IgG1 (mAb-R), which binds to the same epitope as mAb-G but does not exhibit solubility issues. It was observed that addition of salt to mAb-R resulted in relatively weak attractions, while mAb-G showed significantly stronger intermolecular attractions. Two mutants, GM-1 and GM-2, were generated to reduce hydrophobicity of mAb-G. The intermolecular interactions of the mutants were much weaker than those of mAb-G and resembled mAb-R in magnitude. Importantly, both mutants remained soluble at high ionic strength and protein concentration. For all conditions investigated, solubility behavior at high protein concentrations related well to the nature and magnitude of interactions evaluated at lower concentrations. The interaction parameter, kD, determined from DLS was especially valuable in the study of the mutants, as only limited amounts of protein were available and the interaction parameter could be used to predict solubility. K

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

behavior of monoclonal antibody solutions. Mol. Pharmaceutics 2012, 9, 791−802. (32) Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157, 105−132. (33) Adachi, K.; Kim, J.; Konitzer, P.; Asakura, T.; Saviola, B.; Surrey, S. Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers. FEBS Lett. 1993, 315, 47−50. (34) FERREIRA, L.; Schwarz, U.; Keck, W.; Charlier, P.; Dideberg, O.; GHUYSEN, J. Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12. Eur. J. Biochem. 1988, 171, 11−16. (35) Farrell, H. M., Jr.; Kumosinski, T. F.; Pulaski, P.; Thompson, M. P. Calcium-induced associations of the caseins: a thermodynamic linkage approach to precipitation and resolubilization. Arch. Biochem. Biophys. 1988, 265, 146−158. (36) Daugherty, A. L.; Mrsny, R. J. Formulation and delivery issues for monoclonal antibody therapeutics. Adv. Drug Delivery Rev. 2006, 58, 686−706. (37) Sharma, V. K.; Chih, H.; Mrsny, R. J.; Daugherty, A. L. The formulation and delivery of monoclonal antibodies. Therapeutic Monoclonal Antibodies: From Bench to Clinic 2009, 673−709.

(11) Baker, C. M.; Lopes, P. E.; Zhu, X.; Roux, B.; MacKerell, A. D., Jr. Accurate calculation of hydration free energies using pair-specific Lennard-Jones parameters in the CHARMM Drude polarizable force field. J. Chem. Theory Comput. 2010, 6, 1181−1198. (12) Baldwin, R. L. Dynamic hydration shell restores Kauzmann’s 1959 explanation of how the hydrophobic factor drives protein folding. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13052−13056. (13) Valente, J. J.; Payne, R. W.; Manning, M. C.; Wilson, W. W.; Henry, C. S. Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution. Curr. Pharm. Biotechnol. 2005, 6, 427−436. (14) Ruppert, S.; Sandler, S.; Lenhoff, A. Correlation between the osmotic second virial coefficient and the solubility of proteins. Biotechnol. Prog. 2001, 17, 182−187. (15) Schein, C. H. Solubility as a function of protein structure and solvent components. Bio/Technology 1990, 8, 308−317. (16) Arakawa, T.; Timasheff, S. N. [3] Theory of protein solubility. Methods Enzymol. 1985, 114, 49−77. (17) Melander, W.; Horvath, C. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch. Biochem. Biophys. 1977, 183, 200−215. (18) Yadav, S.; Shire, S. J.; Kalonia, D. S. Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J. Pharm. Sci. 2010, 99, 4812−4829. (19) Zhang, T.; Zhang, J.; Hewitt, D.; Tran, B.; Gao, X.; Qiu, Z. J.; Tejada, M.; Gazzano-Santoro, H.; Kao, Y. Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody. Anal. Chem. 2012, 84, 7112−7123. (20) Yadav, S.; Scherer, T. M.; Shire, S. J.; Kalonia, D. S. Use of dynamic light scattering to determine second virial coefficient in a semidilute concentration regime. Anal. Biochem. 2011, 411, 292−296. (21) Brown, W. Dynamic Light Scattering: the Method and Some Applications; Oxford University Press, Oxford, MA, 1993; Vol. 49. (22) Yang, J.; Wang, X.; Fuh, G.; Yu, L.; Wakshull, E.; Khosraviani, M.; Day, E. S.; Demeule, B.; Liu, J.; Shire, S. J. Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A. Mol. Pharmaceutics 2014, 11, 3421−3430. (23) Jiskoot, W.; Crommelin, D. Methods for structural analysis of protein pharmaceuticals. In Analytical Ultracentrifugation; Springer: New York, 2005; Vol. 3, pp 379−412. (24) Lebowitz, J.; Lewis, M. S.; Schuck, P. Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci. 2002, 11, 2067−2079. (25) Shire, S. J. pH-dependent polymerization of a human leukocyte interferon produced by recombinant deoxyribonucleic acid technology. Biochemistry 1983, 22, 2664−2671. (26) Guo, B.; Kao, S.; McDonald, H.; Asanov, A.; Combs, L. L.; William Wilson, W. Correlation of second virial coefficients and solubilities useful in protein crystal growth. J. Cryst. Growth 1999, 196, 424−433. (27) Connolly, B. D.; Petry, C.; Yadav, S.; Demeule, B.; Ciaccio, N.; Moore, J. M.; Shire, S. J.; Gokarn, Y. R. Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter. Biophys. J. 2012, 103, 69−78. (28) Yadav, S.; Liu, J.; Shire, S. J.; Kalonia, D. S. Specific interactions in high concentration antibody solutions resulting in high viscosity. J. Pharm. Sci. 2010, 99, 1152−1168. (29) Sahin, E.; Grillo, A. O.; Perkins, M. D.; Roberts, C. J. Comparative effects of pH and ionic strength on protein−protein interactions, unfolding, and aggregation for IgG1 antibodies. J. Pharm. Sci. 2010, 99, 4830−4848. (30) Saluja, A.; Badkar, A. V.; Zeng, D. L.; Nema, S.; Kalonia, D. S. Ultrasonic storage modulus as a novel parameter for analyzing proteinprotein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements. Biophys. J. 2007, 92, 234−244. (31) Yadav, S.; Laue, T. M.; Kalonia, D. S.; Singh, S. N.; Shire, S. J. The influence of charge distribution on self-association and viscosity L

DOI: 10.1021/acs.molpharmaceut.5b00336 Mol. Pharmaceutics XXXX, XXX, XXX−XXX