Protein Adsorption, Desorption, and Aggregation Mediated by Solid-Liquid Interfaces

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Protein Adsorption, Desorption, and Aggregation Mediated by Solid–Liquid Interfaces TATIANA PEREVOZCHIKOVA,1,2 HIRSH NANDA,2,3 DOUGLAS P. NESTA,4 CHRISTOPHER J. ROBERTS1,2 1

Department of Chemical and Biomolecular Engineering, Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, Delaware 19716 2 Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 3 Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania 4 Department of Biopharmaceutical Product Sciences, GlaxoSmithKline, King of Prussia, Pennsylvania 19406 Received 16 November 2014; revised 19 February 2015; accepted 26 February 2015 Published online 2 April 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24429 ABSTRACT: Adsorption of proteins to solid–fluid interfaces is often empirically found to promote formation of soluble aggregates and larger, subvisible, and visible particles, but key stages in this process are often difficult to probe directly. Aggregation mediated by adsorption to water–silicon oxide (SiOx) interfaces, akin to hydrated glass surfaces, was characterized as a function of pH and ionic strength for alphachymotrypsinogen (aCgn) and for a monoclonal antibody (IgG1). A flow cell permitted neutron reflectivity for protein layers adsorbed to clean SiOx surfaces, as well as after successive “rinse” steps. Aggregates recovered in solution after gently “rinsing” the surface were characterized by neutron scattering, microscopy, and fluorescence spectroscopy. IgG1 molecules oriented primarily “flat” against the SiOx surface, with the primary protein layer desorbed to a minimal extent, whereas a diffuse overlayer was easily rinsed off. aCgn molecules were resistant to desorption when they appeared to be unfolded at the interface, but were otherwise easily removed. For cases where strong binding occurred, protein that did desorb was a mixture of monomer and small amounts of HMW aggregates (for aCgn) or subvisible particles (for IgG1). Changes in adsorption and/or unfolding with pH indicated that electrostatic interactions were important in all cases.  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1946–1959, 2015 Keywords: adsorption; desorption; neutron reflectivity; scattering; stability; protein aggregation; particle sizing

INTRODUCTION Aggregation of protein-based therapeutic products is a common issue during product development and multiple stages in manufacturing, as well as during subsequent transportation and storage.1,2 Protein aggregation mediated by partial unfolding can be accelerated by deliberate or inadvertent stresses, such as agitation, exposure to elevated temperatures, chemical degradation, and exposure to solid–liquid and air–water interfaces.3 Surface-mediated unfolding is a potential issue during manufacturing and storage, making it an important consideration of formulation and process development strategies.4 Association of proteins at bulk interfaces has been empirically implicated in aggregate formation for some proteins, but the mechanistic details of the process are not fully known, at least in part because the large majority of characterization techniques measure protein structure and aggregation state only in bulk solution.2,5,6 The current understanding is that aggregation because of adsorption/desorption of misfolded protein from the surface may occur as a result of two scenarios. First, it may originate from aggregates that are formed on the surfaces and subseCorrespondence to: Christopher J. Roberts (Telephone: +302-831-0838; Fax: +302-831-1048; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Tatiana Perevozchikova’s present address is Department of Biopharmaceutical Product Sciences, GlaxoSmithKline, King of Prussia, Pennsylvania. Hirsh Nanda’s present address is Janssen Research & Development, Johnson & Johnson, Spring House, PA. Journal of Pharmaceutical Sciences, Vol. 104, 1946–1959 (2015)

 C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

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quently desorbed into solution. Alternatively or additionally, it can be initiated by unfolded monomeric protein that aggregates rapidly in solution after desorption.5,6 The existence of small aggregates that are capable of seeding further aggregation, as well as the presence of large aggregates desorbed directly from surfaces, are both of concern because of their potential for triggering unwanted immunogenic responses in patients.7 Although many open questions remain regarding the nature of immunogenicity as it relates to protein aggregates, the need for characterization and control of aggregation/particle formation is widely recognized.8–11 Previous studies have considered different types of stresses that therapeutic proteins may be subjected to via exposure to industrially relevant surfaces. It was previously shown that different agitation rates and surface roughness characteristics are capable of inducing variable amount of detectable aggregates in protein-containing solutions.5,6 However, experiments assessing the structure of interfacial layers formed on these surfaces were beyond the scope of many previous studies. In general, detailed understanding of the relationship between the formation of aggregates and protein surface association remains incomplete. A number of theoretical and experimental studies have explored the conformation of protein biofilms on various surfaces.12–14 However, the primary focus of most research has been on changes in protein conformation and/or ligand binding for proteins that remain physically or chemically bound to the surface, rather than assessing subsequent aggregate formation and the presence of detectable aggregates in bulk solution. Previous work suggests that adsorption of proteins to hydrophilic

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RESEARCH ARTICLE – Pharmaceutical Biotechnology

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MATERIALS AND METHODS aCgn Solution Preparation

Figure 1. The structural dimensions of aCgn and IgG1 proteins. (a) The crystal structure of aCgn (PDB file 1HCH) demonstrates an ˚ diameter. (b–c) Potential orientaoverall spherical shape with 40 A tions of IgG1 antibody relative to a surface. (b) The flat-on orientation ˚ in height. (c) Placing of the IgG1 molecule in the of an antibody is 55 A ˚ (d) The end-on orientation side-on position results in a height of 105 A. ˚ has a maximum length of 125 A.

surfaces is primarily dictated by electrostatic surface–protein interactions, whereas the prevalence of nonpolar patches on the surface of a protein is more important in determining adsorption to hydrophobic surfaces.15–22 The present work focuses on aggregation mediated by adsorption to aqueous interfaces with silicon oxide (SiOx) for two model proteins: a relatively small (26 kDa) globular protein, alpha-chymotrypsinogen (aCgn); and a larger (150 kDa), multidomain immunoglobulin gamma-1 (IgG1) (Fig. 1). SiOx, a primary component of glass vials used for biopharmaceutical products and/or manufacturing, was chosen to mimic the walls of vials. By using this material, it is also relatively straightforward to obtain sufficiently smooth and flat surfaces that permit high-resolution NR measurements for adsorption of proteins or similarly sized nanoparticles.5,16,21,23–26 The packing density and surface thickness of adsorbed proteins was determined as a function of solution pH and ionic strength, as well as changes in the surface layer(s) upon gentle rinsing with buffer solution. Desorption of the proteins from SiOx was initiated by a series of buffer rinses and followed by observing the conformational changes in the adsorbed layer, as well as the presence of aggregates in the rinse solution. Previous work demonstrated that both aCgn and this IgG1 form aggregates when heated in bulk solution, ranging from small oligomers to large (micron sized) particles.27–33 This study also compared and contrasted the aggregate properties and aggregation mechanisms for these two proteins when stressed by heating (previous work),34 versus exposure to aqueous SiOx interfaces (present work.) The results of this work indicate that electrostatic forces play an important role in mediating the adsorption and conformational changes of both proteins at the SiOx interface. The results also suggest links between the strength and likelihood of attractive protein–surface and protein–protein interactions, the tendency for proteins to unfold at the interface, aggregation as a result of protein unfolding, and the relative ease with which adsorption is reversed. DOI 10.1002/jps.24429

Alpha-chymotrypsinogen A type II from bovine pancreas (crystallized and lyophilized, product number C4879) was purchased form Sigma–Aldrich (St. Louis, Missouri), and used without further purification. Protein stock solutions were prepared gravimetrically at a concentration of 15 mg/mL, and triple dialyzed against 30 mM citrate buffer at pH 3.5 or 4.5 to remove the residual salt content from the lyophilized powder. Each dialysis step used a 10-fold volume of buffer relative to the volume of protein stock solution, and used cassettes with a 10-kDa molecular weight cutoff (ThermoScientific Slide-A-LyzerTM ), with a minimum of 24 h for each dialysis step. Citrate buffers were prepared from combinations of sodium citrate dihydrate (Aldrich, Milwaukee, Wisconsin) and citric acid (Sigma–Aldrich), depending on the desired pH. The pH of all solutions was measured to ensure that the actual values agreed with the target values. Two forms of the buffer were used for neutron reflectivity (NR) and small-angle neutron scattering (SANS) studies: the hydrogenated form, prepared with Milli-Q filtered water (Millipore, Billerica, Massachusetts) as a solvent; and the deuterated form combined with D2 O (Cambridge Isotope Laboratories, Tewksbury, Massachusetts). After dialysis, all solutions were refrigerated and protected from light exposure, and used within 2 days of preparation. Protein concentrations were verified using UV/Vis spectrophotometry with previously reported molar absorbtivity values.27 mAb Solution Preparation Purified IgG1 antibody was provided by GlaxoSmithKline as a stock solution with a protein concentration of 29 mg/mL. Stock protein solutions were dialyzed into deuterated or hydrogenated buffers at pH 4.5 and 6.2. Buffer solutions were prepared according to the procedure described above. After dialysis, protein solutions were diluted gravimetrically to 15 mg/mL (concentration confirmed by UV/Vis absorbance), and stored as described above. NR Measurements Substrate Preparation Silicon wafers of approximately 7.6 mm diameter, 5000 :m ˚ thick n-type Si:P[100] with one side polished to less than 5 A roughness, were purchased from El-Cat (Waldwick, New Jersey). Prior to experiments, Si wafers were prepared by soaking in Piranha etch solution (3:1 concentrated sulfuric acid:30% hydrogen peroxide) for 30 min. Wafers were then thoroughly rinsed in ultrapure Milli-Q water before assembly into wet-cells that were specially designed for laminar flow NIST reflectometry. This treatment consistently resulted in a contaminant-free, native SiOx layer on the wafer, as confirmed by NR prior to exposure to protein-containing solutions (see next subsection). Neutron Reflectivity Neutron reflectivity measurements were performed on the NG7 horizontal reflectometer at the NIST Center for Neutron Scattering. A momentum transfer, qz , range between 0.008 and ˚ −1 was accessed in most measurements. Typical mea0.250 A surements used two solvent isotopic contrasts, consisting of aqueous buffer prepared in either 100% D2 O or H2 O. For each contrast, 6 h of data collection provided sufficient counting

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RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 2. Neutron reflectivity measurement geometry and schematic of the NR flow cell. A protein solution is introduced to the NR flow cell through the inlet tubing and collected on the opposite side of the cell. A space for a protein solution is provided by a thin Viton spacer and can be adjusted depending on the objective of the study (5). Adsorption is occurring on the SiOx surface (2) and studied by NR. After the structure of the initial adsorbed protein phase (3) is characterized, the formed layer is exposed to a series of buffer rinses in a desorption step of a study. In a NR experiment, a flat collimated neutron beam is entering through the silicon sample wafer (1), coated with the native SiOx layer (2). The neutrons reflect from the various interfaces present in the cell: Si wafer–SiOx (1 and 2), SiOx–protein (2 and 3) and protein solvent (3 and 4). The constructive and destructive neutron wave interference gives a rise to a reflectivity profile, describing density, width, and a roughness of the layers present on the surface.

statistics to resolve signal over background counts with counting times weighted toward high q region of the scan. The flowthrough cell design allows for in situ buffer/sample exchange on the instrument. A schematic of the flow-cell setup and a description of the neutron beam reflection geometry are provided in Figure 2. At the start of each NR experiment, the flow-cell was filled with buffer (no protein present) and tested for cleanliness of the SiOx surface with NR. For each protein and buffer condition that was tested, 3 mL of protein solution (twice the flowcell volume) was then allowed to flow slowly through the cell, with the cell completely filled with liquid at all times. A flow rate of 6 mL/h was applied using a syringe pump to introduce the solution through a 100-:m gap created by a viton gasket at the inlet. The excess solution (1.5 mL) was collected from the outlet and labeled as “after reflectivity cell” to indicate the solution had contacted the flow-cell, but for only a short period of time. The remaining solution in the flow-cell (1.5 mL) was incubated for 6 h to allow for protein adsorption and any subsequent changes in the layer(s) of protein at the surface. Reflectivity spectra were collected throughout the 6-h period. After the 6-h holding period, a 6-mL aliquot of buffer solution (same pH/salt concentration as the corresponding protein solution) was passed through the cell at a flow rate of 12 mL/h and collected in two equal fractions designated “buffer wash 1” (BW1) and “buffer wash 2” (BW2), respectively. After the 6-mL buffer rinse, NR spectra were collected on the residual protein layer(s) at the SiOx interface. Data Analysis Analysis of NR data was performed by fitting a model of the spatial distribution of the protein mass, normal to the

SiOx surface (“z” axis in Fig. 2), to the measured reflectivity spectra. A Hermite spline was used to model the distribution of the protein along the z-axis (Fig. 2) without any assumptions of the protein profile other than that it was uniform across the surface (i.e., the “x” and “y” directions on the SiOx surface). The thickness and roughness of the native SiOx layer were also free parameters in the model, whereas the SLD was fixed to the ideal value of bulk SiOx of 2.07e˚ −2 . Fitting was performed with the Refl1D software package 6A (http://www.reflectometry.org/danse/docs/refl1d/index.html) and the molgroups extension that provides a compositionspace model for fitting the underlying z-dimension spatial distribution of adsorbed protein.35 Reflectivity spectra were collected using two isotopic contrast aqueous buffers consisting of H2 O and D2 O. In all models, the protein is assumed to have a constant nSLD along the membrane normal direction. A representative value for proteins was determined by taking tabulated amino acid volumes and typical amino acid frequen˚ −2 in cies in proteins resulting in a nSLD value of 2.23e-6 A −2 ˚ H2 O and 3.14e-6 A in D2 O assuming full exchange of all labile hydrogens.36 Provided SLD values for the buffer conditions, reflectivity profiles are calculated from a single protein distribution model for both H2 O and D2 O solvent contrasts and fit simultaneously to the respective experimental datasets. A Monte Carlo error analysis procedure was used to determine the confidence intervals in the protein profile.37 Solution Structure Alpha-chymotrypsinogen and IgG1 were characterized in bulk solution prior to, and after adsorption/desorption to the SiOx surface, using SANS, Thioflavin T (ThT) binding, and microflow imaging (MFI). Specifically, the following four sample types were collected and tested as part of the flow-cell experiments. The first set of samples was the stock solution of a given protein and pH/salt conditions, which had never been applied to the SiOx surface. The second was an “after reflectivity cell” solution as described above (hereafter referred to as AR), consisting of the sample collected directly after the flow-through NR cell with a short contact time with the surface. And finally, “BW1” and “BW2”, which were obtained from rinsing the formed layers with the corresponding buffers. The resulting protein concentration in each of the samples varied significantly because of the loss of protein to the surface and dilution with buffers during desorption steps. The protein concentrations obtained in AR, BW1, and BW2 aliquots were measured by UV adsorption at 280 nm, and are tabulated as a part of the results. Small-Angle Neutron Scattering The protein samples collected from NR experiments were characterized by SANS to assess measurable changes in protein structure and the presence of aggregates. Only the aliquots in deuterated citrate buffers were used for SANS, as D2 Obased buffers have a much smaller incoherent background and enhance the contrast between the protein and solvent. The SANS data were collected on the NG-7 and NG-3 beamlines at the NIST National Center for Neutron Research located in Gaithersburg, Maryland. The samples were placed in demountable 1-mm pathlength titanium cells with quartz windows and a final protein volume of 0.5 mL. To obtain information on both the form factor of the proteins and interparticle interactions, all samples were measured at three sample-to-detector distances:

Perevozchikova et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1946–1959, 2015

DOI 10.1002/jps.24429

RESEARCH ARTICLE – Pharmaceutical Biotechnology

˚ wavelength neutrons. On the ba1, 4, and 13 m utilizing 6 A sis of these instrument configurations, the magnitude of the wavevector transfer, Q = 4B sin (2), where 22 is the scatter˚ −1 . Data collection ing angle, was in the range of 0.006–0.5 A times varied depending on the detector position and the sample to be measured, but were chosen to provide a sufficient signal count over the background. Data reduction was performed using Igor Pro software (WaveMetrics, Lake Oswego, Oregon) with the NCNR SANS macros using the standard procedure.38 The collected data were corrected for detector background and sensitivity, as well as the scattering contribution from empty cells. The protein scattering profiles were normalized by incident beam flux and the raw intensities were placed on absolute scale using direct beam measurements. The Q-independent excess incoherent signal was also subtracted as a background from all SANS profiles. ThT Fluorescence Because of the high sensitivity of the fluorescence measurements and the optimal amount of particles reliably detectable by MFI, the samples collected during NR experiments were diluted to the values indicated in Table 1. The potentially amyloid-like structural features of each protein solution were probed using ThT dye (MP Biomedicals, Solon, Ohio). ThT stock solution was prepared by dissolving the dye into Milli-Q water to a final concentration of 2 mg/mL. The binding of ThT to aggregates was assessed by diluting ThT stock into a given protein sample to a 15-fold molar excess of the dye over the protein concentration. The ThT fluorescence signal was measured using a spectrofluorimeter (FluoroLog, HORIBA Jobin Yvon, New Jersey) with an excitation wavelength of 450 nm and emission wavelength range of 460–600 nm. Two ThT measurements were performed and averaged for every dataset and each reported spectrum was corrected for the differences in concentration of the collected sample versus the corresponding stock solution, with the stock solutions (unexposed to the SiOx surface) showing negligible ThT fluorescence (not shown). Intrinsic Fluorescence Spectroscopy Unfolding and misfolding in the proteins’ tertiary structure as a result of adsorption/desorption was examined by steady-state intrinsic tryptophan fluorescence. The protein solutions were prepared as described above (stock solutions, AR, BW1, BW2) and the corresponding intrinsic fluorescence spectra were obtained on the spectrofluorimeter described above, with an ex-

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citation length of 295 nm and emission wavelength range between 300 and 450 nm. Each reported spectrum is an average of two measurements with the baseline obtained from the fluorescence signal of the buffer subtracted from the sample. Particle Measurements by MFI A Brightwell (Ottawa, Ontario, Canada) MFI 4100 instrument was used for counting and characterizing particles in the aliquots collected during NR experiments and diluted as described above. The samples were collected into PETG-certified clean containers (Nalgene; Thermo Fischer Scientific) to assure minimal artifacts from foreign particulates because of the choice of container/closure. The protein samples and corresponding buffers were left to stand at room temperature for approximately 10 min to remove any air bubbles present in the solution. The resulting solutions were analyzed with the instrument configuration in “set point #” mode, optimized for a maximum magnification and a minimum particle dimension of 2 :m. Prior to each sample measurement, 0.6 mL of a sample was used to optimize the illumination for each measurement, followed by 0.8 mL of protein solution analyzed. The buffers were also evaluated to assess their contribution to the sample particle profiles and were used as a negative control. The amount of protein incorporated into detected particles was evaluated as previously described.39

RESULTS Adsorption/Desorption Behavior of aCgn at SiOx–Water Interface The adsorption behavior of aCgn to a SiOx surface was studied as a function of solution pH and NaCl concentration. Protein solutions at 15 mg/mL were prepared at pH 3.5 and pH 4.5 and flowed through the NR flow-cell as depicted in Figure 2 and described in Materials and Methods. Measured NR patterns of the adsorbed protein in H2 O and D2 O buffer contrast are shown in Supplementary Material (Fig. S1). The patterns were interpreted by fitting a model that provides volume fraction of protein as a function of distance from the SiOx surface (see Materials and Methods). Figure 3a shows the best-fit volume fraction profile of aCgn at pH 3.5. The protein layer begins at the SiOx surface (denoted as the x-axis zero position) and has ˚ The maximum volume fraction a width of approximately 50 A. after 3 h of incubation is approximately 0.38 and increases to 0.42 after an additional 3 h of incubation to allow protein to adsorb to the SiOx surface. Rinsing the flow-cell with 6 mL of

Table 1. Immunoglobulin Gamma-1 Solution Properties Studied by MFI Sample

Concentration Analyzed (mg/mL)

IgG 1 pH 4.5 30 mM Citrate Stock 0.26 AR 0.15 BW1 0.13 BW2 0.028

Particle Concentration (Particle/mL)

ECD Range (:m)

Percent Protein Mass in Particles

Percent Small Particles by Count

Percent Protein in Large Particles by Mass

2561 2951 3198 2260

1–23 1–46 1–52 1–62

0.03 0.40 0.60 3.34

99 95 96 94

33 78 91 99

1–56 1–65 1–99 1–91

0.65 2.74 1.23 3.92

97 91 96 95

94 96 95 99

IgG pH 4.5 100 mM NaCl 30 mM Citrate Stock 0.29 8890 AR 0.11 4150 BW1 0.46 15,254 BW2 0.028 661

DOI 10.1002/jps.24429

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RESEARCH ARTICLE – Pharmaceutical Biotechnology

x, y, and z coordinates of each atom provided by the resolved protein structure (PDB file 1HCH), a volume fraction profile was calculated along a single axis direction and overlaid on the NR profiles in Figure 3. At pH 3.5, the adsorbed protein layer from NR is wider than that predicted from a simple monolayer calculated from the X-ray crystal structure. The experimental profile is not sufficiently wide to suggest bilayer or multilayer adsorption. Therefore, it is speculated that some degree of conformational change/unfolding has occurred for proteins on the surface. In contrast, the calculated profile based on the X-ray crystal structure is in good agreement with NR results at pH 4.5, suggesting the protein average structure remains closer to that of folded conformation than at pH 3.5. Because of the relatively isotropic nature of the aCgn PDB structure, the 1-D calculated density profiles in Figure 3 was reasonably similar even when rotated to randomize the axial direction around which the profile was calculated (not shown). It should be noted that NR necessarily averages laterally with respect to the SiOx surface, and therefore one cannot infer information about spatial heterogeneity parallel to the SiOx surface without the use of information from other techniques (e.g., surface imaging). Solution Properties of aCgn as a Consequence of Surface Adsorption/Desorption

Figure 3. Neutron reflectivity profile on adsorption of aCgn at different pH conditions. Y-axis demonstrates the volume fraction of the molecule within a layer; X-axis shows the angstrom distance from SiOx interface. (a) The structure of the aCgn formed within 3 and 6 h and after the buffer wash is shown by blue, red, and green curve, respectively. The black line demonstrates the calculated NR profile of a layer, which would result from placing an X-ray structure of the aCgn (PDB file 1HCH) in a monolayer fashion. (b) The comparison of the layer formed at pH 3.5 (red curve) and pH 4.5 (blue curve) with the X-ray structure layer scaled for the best fit.

buffer solution resulted in partial desorption, with a residual protein layer of essentially the same width but a maximum volume fraction of 0.1. At pH 4.5, the adsorption/desorption behavior of aCgn was markedly different. The protein profile ˚ and a maxafter adsorption had a width of approximately 40 A imum volume fraction of approximately 0.2 that was invariant after the first 3 h of incubation. Subsequent rinsing with buffer solution resulted in completely removing the protein layer (Fig. 3b). It is possible to qualitatively evaluate the conformational state of the adsorbed protein, to a limited degree, by comparing the shape of the volume fraction profiles (i.e., volume fraction or packing density as a function of distance from the SiOx plane in Fig. 3) to the known X-ray crystal structure of aCgn. From the

Small-angle neutron scattering can detect ensemble averaged structural changes on length scales ranging from approxi˚ In order to test whether mately 10 to approximately 1000 A. exposure of aCgn to the SiOx surface resulted in protein with a perturbed structure once desorbed to the bulk solution, aliquots of the protein solution were collected during different stages of the NR experiment at both pH values. Figure 4a annotates the different regimes of an illustrative SANS profile and specifies approximately which ranges of the scattering vector correspond to information on protein conformation or protein–protein interactions and assembly into aggregates, for the typical dimensions of the proteins considered here (note that the x axis is on a logarithmic scale). The SANS results for pH 3.5 are shown for the stock protein solution, after-reflectivity-cell (AR) fraction and BW1 and BW2 fractions (Fig. 4a). The SANS curves for all datasets exhibit similar shapes at the high Q regime ˚ −1 ), and indicates no de(scattering vector range of 0.06–0.35 A tectable changes in the intramolecular conformation of aCgn for all samples. However, an upturn at the low-Q regime (0.008– ˚ −1 ) for buffer wash samples (BW1 and BW2) signifies the 0.06 A presence of larger aggregates in the solution. It is difficult to reliably quantify the molecular weight of the aggregates, as the data did not reach sufficiently low Q to become independent of Q, and because the scattering profile is a combination of both monomers and a presumably polydisperse mixture of aggregates at an unknown concentration. However, MFI measurements (data not shown) showed no detectable particles relative to buffer controls, and this therefore restricted the size range of the aggregates to approximately 0.06–1 :m. Small-angle neutron scattering profiles for the collected fractions at pH 4.5 are shown in Figure 4b. In this case, SANS profiles were identical for all collected fractions with only a shift in intensity because of the difference in protein concentration. Thus, there is no indication of conformational change or the formation of aggregates in the desorbed species at pH 4.5. Larger aggregates, on the order of micron or larger length scales, were also not observed by MFI (data not shown). Analogous

Perevozchikova et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:1946–1959, 2015

DOI 10.1002/jps.24429

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 4. Aggregation behavior of aCgn in solution for both pH observed by SANS. (a) aCgn at pH 3.5: red open circles—stock solution, black solid circles—“after reflectivity,” green and blue solid circles— BW1 and BW2, respectively. The upturn at the low Q regime on data collected from buffer washes indicates the presence of aggregates. (b) aCgn at pH 4.2: red solid circles—stock solution, green circles— “after reflectivity,” black circles—BW1. No upturn at the low Q is observed at either of the scattering profiles.

measurements were performed for aCgn at higher ionic strength (with 100 mM NaCl added) for pH 3.5 and 4.5, but the results were essentially indistinguishable from those described above (data not shown). Adsorption/Desorption Behavior of IgG1 on SiOx Analogous adsorption/desorption experiments for the monoclonal antibody were performed with pH 4.5 and pH 6.2 solutions. As noted earlier, aCgn and IgG1 have significantly different physical properties and aggregation mechanisms in solution as a function of pH. Therefore, simply choosing the same pH values for all cases was unlikely to be useful when placed within the broader context of their overall aggregation behavior. As such, these pH values were chosen because the mechanisms by which IgG1 aggregates grow to larger sizes in bulk solution at these conditions are similar to those for aCgn at pH 3.5 and 4.5, respectively. At pH 4.5 (IgG1) and pH 3.5 (aCgn), DOI 10.1002/jps.24429

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Figure 5. pH dependence for NR profile after adsorption and desorption of the IgG1. (a) pH 4.5: after initial adsorption step (solid green line), and after a desorption step/buffer wash (solid yellow line). Insert: dependence of NR profiles on IgG1 concentration (see also, figure legend). (b) pH 6.2: after initial adsorption step (solid blue), and desorption step/buffer wash (solid red). The dotted lines for each color represent the maxima and minima of the confidence intervals (calculated as 2 sigma) for the corresponding NR profiles given as solid lines.

the mechanism of aggregate growth is predominantly monomer addition (i.e., a chain polymerization pathway), and aggregates remain in solution and are easily detected and quantified with chromatography and small-angle scattering, whereas at the higher pH values for both proteins, the growth mechanism in bulk solution is predominantly aggregate–aggregate coalescence and ultimately macroscopic phase separation in the form of visible particles and turbid suspensions.28,29 For both proteins, conformational stability increases significantly from lower-pH to higher-pH conditions tested here.27–29 Figure 5a shows the experimental NR profiles IgG1 solutions exposed to the SiOx surface at pH 4.5. For the profile after 3 h of stagnant incubation to allow adsorption (without rinsing), there is a peak proximal to the SiOx surface with a maximum volume fraction of 0.44 and a width of approximately ˚ (estimated by fitting a Gaussian peak shape to the profile). 65 A The volume fraction profile also shows a tailing distribution of protein mass extending away SiOx interface and into the

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RESEARCH ARTICLE – Pharmaceutical Biotechnology

Table 2. The Characteristics of IgG1 Layers Formed by Adsorption to SiOx and Desorption with the Buffers at the Target pH and Ionic Strength Stock Sample aCgn, pH 3.5 aCgn, pH 4.5 IgG, pH 4.5 IgG, pH 6.2 IgG, pH 4.5 + salt IgG, pH 6.2 + salt

Integrated Surface Area upon Adsorption

Integrated Surface Area upon Desorption

Fraction of Surface Cross-Section

Estimated Maximum Fraction of Surface Coverage

Percent of the Estimated Maximum Surface Coverage

14.08 3.95 20.73 29.88 16.53 18.68

4.46 0 17.39 18.36 13.16 11.07

0.42 0.33 0.43 0.51 0.39 0.38

0.24 0.20 0.57 0.62 0.69 0.71

175 61 75 80 56 53

bulk aqueous phase. The observed width of the main peak suggests a flat-on conformation of monoclonal antibody in the contact with SiOx surface (cf., dimensions from IgG1 structure in Fig. 1), assuming the majority of the adsorbed proteins adopt a similar orientation. This is in an agreement with previous studies of adsorption of monoclonal antibodies.15,16,18 Longer incubation times (above 3 h) did not result in observable changes of IgG1 layer (data not shown). After a desorption step consisting of 6 mL of buffer rinsing, the IgG layer remained largely intact (Fig. 5a, orange curve). The most notable difference was the removal of the trailing portion of NR profile, suggesting that contribution is likely because of diffusely packed and/or weakly associated protein molecules. Interestingly, exposure of the surface to the protein at significantly lower protein concentration (0.2 mg/mL) resulted in a NR profile that was very similar to that for the higher protein concentration, with the only difference being a lack of the distal “tail” (Fig. 5a, insert). This observation is in contrast to the results published by Xu et al.,16 in which IgG adsorption was studied at much lower concentration (0.002–0.05 mg/mL) and the increase in concentration from 0.002 to 0.01 mg/mL resulted in significantly different profile for the adsorbed layer(s). To assess the changes in the amount of protein at the surface after adsorption and desorption steps, the volume fraction profiles were integrated over the distance normal to the SiOx surface, resulting in a quantity that provides a measure of the total amount of protein, per unit area, on the surface. Table 2 shows that for IgG1 at pH 4.5, over 80% of the protein material remains after the buffer rinse, with a majority of the protein loss attributed to the distal protein layer(s) and possibly a small amount of the more tightly surface-associated protein molecules. It is not possible to determine from these experiments whether IgG desorbed and readsorbed during a given rinse step, although one might speculate such readsorption could result in a more strongly adsorbed IgG configuration (e.g., flat-on, to provide more points of contact between the surface of the IgG molecule(s) and the SiOx substrate). Adsorption of IgG1 at a pH 6.2 resulted in the formation of a protein layer with a peak volume fraction of approximately 0.51 (Fig. 5b). The profile is highly asymmetric, with a broad shoulder on the distal side to the SiOx interface. Fitting the main ˚ the shoulder peak to a Gaussian function gives a width of 64 A; is not accounted for by the Gaussian fit. One possible interpretation of the additional protein material at larger distances from the SiOx surface is that one of the IgG domains protrudes from the surface and thus extends the protein distribution from a purely flat-on conformation (see Fig. 1b). Alternatively, the asymmetric peak can be well approximated by two Gaussian ˚ from the SiOx interface with distributions, one centered at 31 A

˚ and a broader peak centered at a width of approximately 64 A ˚ with a width of 105 A, ˚ each peak of approximately equal 59 A area. This suggests a second possible interpretation is the coexistence of two IgG orientations, a narrow, flat-on (Fig. 1b) and a wider side-on (Fig. 1c). However it is difficult to distinguish between the two interpretations based on a composite structural profile that NR provides. Given the limits of NR, one should restrict interpretations of such profiles to only qualitative and semiquantitative aspects of the protein film “structure.” Specific deuteration of Fc or Fab domains may provide a clearer picture of antibody organization at the interface, suggesting some directions for future work. Rinsing the initially formed layer resulted in an overall reduction of the protein volume fraction, and removal of the extended, diffuse/distal protein layer (Fig. 5b). Integration of the surface coverage for both the adsorption and desorption steps shows a 40% reduction in total protein volume after rinsing (cf., Table 2). The width of the protein NR profile also suggests an organization of IgG mass at the surface that may be primarily, but not fully, described by a compact, flat-on orientation. However, as noted above, more structurally detailed interpretation of the distribution of protein domains at the surface would require additional measurements with specific domain labeling, as well as possibly imaging techniques to discriminate between relatively uniform versus “patchy” films that were beyond the scope of this work. Comparison with Higher Ionic Strength Conditions Solution pH has a large effect on the net charge of the IgG1 protein in this study. For example, at pH 4.5, the antibody has a +15 experimental net charge in these buffer conditions, whereas at pH 5.5, the experimental net charge reduces to +5.34 Although the net charge was not measured at pH 6.2, it is anticipated to be small and positive based on earlier calculations that accounted for the amino acid composition of titratable side chains.34 At the acidic pH values used here, SiOx is expected to have a net negative surface charge density at the aqueous interface.40 One may expect strong electrostatic attractions between the oppositely charged protein and SiOx surface. If that were the only factor driving protein adsorption, then adding salt could, in principle, screen electrostatic attractions and therefore greatly alter the net adsorption and structure of the IgG1 layer. Immunoglobulin gamma-1 solutions were exposed to fresh SiOx surfaces at a higher ionic strength (buffer plus 100 mM added NaCl) for the same pH conditions described above. The results of the NR experiments for the adsorption behavior of IgG1 in high salt conditions are shown in Figure 6. At pH 4.5,

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age of the protein in terms of a volume fraction. Table 2 shows the fractional surface coverage of IgG1 at pH 4.5 is approximately 0.43 at buffer-only conditions and is approximately 0.42 at higher salt conditions. The corresponding values at pH 6.2 are approximately 0.51 and 0.38, respectively. It was then estimated how close these values are to a theoretical maximum for packing of an IgG1 monolayer on the SiOx surface. For this estimate, it was assumed that the effective protein radius was the sum of its hydrodynamic radius and the characteristic charge screening length, so as to roughly account for both steric and electrostatic repulsions that presumably limit the minimum center-to-center distance between proteins adsorbed at the surface. The low-salt buffer-only solution had ionic strength values of 50 mM at pH 4.5, and 90 mM at pH 6.2; and the high-salt buffer had ionic strength values of 150 mM at pH 4.5, and 190 mM at pH 6.2. The calculated screening length was 1.7 and 0.84 nm, respectively, with the simplifying assumptions of a Debye screening model and a 1:1 electrolyte. The hydrodynamic radius of this IgG1 was determined previously to be 5.2 nm.30 The maximum possible surface packing density, Dmax , depends on the ratio of the areas determined by the hydrodynamic radius, Ah , and the area because of the effective radius, Aeff , such that: Dmax =

Figure 6. Neutron reflectivity profiles for adsorption/desorption of IgG1 at higher ionic strength (30 mM citrate, 100 mM added NaCl). (a) pH 4.5 after initial adsorption (green line) and subsequent rinse step (yellow line). (b) pH 6.2 after initial adsorption (purple line) and after subsequent rinsing (red line). Dashed lines are confidence intervals, as in Figure 5.

the experimentally derived volume fraction profiles were very similar to the low salt condition. At high salt, the maximum protein volume fraction reached approximately 0.42 and the ˚ After buffer rinse, 80% peak had a width of approximately 50 A. of the protein layer remained (Table 2). The addition of high salt had a larger effect at pH 6.2 where the peak volume fraction was approximately 0.39 for the initially adsorbed layer, which was a 20% reduction from the lower salt condition. Similar to the buffer-only condition, a broader asymmetrical peak was observed compared with its pH 4.5 counterpart. However, in the higher salt case, the distribution is not well approximated by fitting two peaks of different widths. Fitting to a Gaussian ˚ for the adsorption curve distribution gives a peak width of 74 A ˚ for the desorption curve. The buffer rinse left 60% and 66 A of the original protein layer, thereby removing a significantly greater fraction of protein than in the buffer-only case (See Fig. 6; Table 2). IgG1 Surface Coverage The profiles in Figures 5 and 6 demonstrate a maximum peak that can be interpreted as the maximum SiOx surface coverDOI 10.1002/jps.24429

Ah × 0.91 Aeff

where the 0.91 factor comes from the highest packing density achievable by equally sized circles on a 2-D plane. Thus, for IgG1, the Dmax was calculated to be 0.51 and 0.67 for low-salt and high-salt conditions, respectively. On the basis of the experimental surface coverage listed in Table 2, at low salt, the IgG1 displays close (89%) to its estimated maximum surface coverage at pH 4.5, and even closer (98%) to the estimated maximum at pH 6.2. At high salt, the IgG1 molecules can theoretically pack much closer because of the shorter screening length, but the lower experimental values result in achieving only approximately 50% of the maximum possible surface packing. The particular numbers above are only estimates, based on the simplifying assumptions in performing the calculations. Despite that limitation, the analysis indicates that the surface layer of protein is at least reasonably approximated as a wellpacked monolayer when the attractions between protein and surface are large. Solution Characterization of IgG1 After Adsorption/Desorption Aliquots of IgG1 solution were collected during the progress of NR adsorption/desorption experiments as described in Materials and Methods. To assess changes in the conformational state and the formation of submicron aggregates of IgG1 in solution, the collected samples were characterized first with SANS. The SANS profiles for each pH condition are shown in Supplemental Figures S2A and S2B, and are essentially unchanged between the different samples. This indicates that all IgG1 molecules in solution are effectively in folded conformations, and does not show aggregation that is detectable by SANS. The drop in the scattering intensity between the “after reflectivity” fractions and the “BW1” solution is attributed to the loss of the protein to the surface, and dilution with the buffer during the rinse step. The SANS data contain information only on “BW1,” as the concentration of the protein in the subsequent rinse,

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“buffer wash 2,” was too low for the sensitivity limit of SANS (not shown). The “buffer wash 2” sample, however, was additionally characterized because it contained only protein that desorbed from the relatively strongly bound surface layer, whereas the first wash likely contained a high fraction of weakly bound protein species. To assess structural characteristics of protein species desorbed during “buffer wash 2,” other aggregation-sensitive, low-concentration techniques were employed—specifically, ThT binding, intrinsic fluorescence spectroscopy, and MFI. The lattermost technique, in particular, enables probing of small concentrations of aggregates with sizes in the range of microns, and to obtain quantitative information regarding the particle concentration(s) in bulk solution. Surface Adsorption Leading to Particle Formation for IgG1 The amount and morphology of large particles/aggregates was assessed by MFI. At pH 6.2, the formation of smaller, soluble aggregates was not detected by any of the techniques used in this study. In previous work, it was shown for this IgG1 molecule that the formation of macroscopic, insoluble aggregates is prevalent at pH values near and above 5.5.29,34 Such large aggregates readily sediment from solution and are beyond the characteristic lengths probed by either SANS or MFI. Therefore, the results reported here only focus on particle formation and aggregate characterization for the pH 4.5 solution conditions. As previously described, the total protein concentration in collected aliquots of IgG1 solutions from the reflectivity cell steadily decreased because of dilution by the rinsing buffer. Therefore, the evaluation of these samples solely by particle counts per unit volume would be insufficient to compare the amount of protein aggregated during adsorption/desorption steps. Table 1 shows the concentrations of illustrative IgG samples used for subsequent solution analysis (MFI, intrinsic, and ThT fluorescence). To evaluate the progression of particle formation, the total mass of protein (per unit volume of solution) incorporated into aggregates was calculated using a previously described method,39 and compared with the overall concentration of recovered protein in a given aliquot. The two key parameters that served as the basis for calculating the protein mass that was incorporated into particles were the equivalent circular diameter (ECD) and the total particle concentration (see Materials and Methods).39 As shown in Figure 7a, the stock solution of IgG1 at pH 4.5 has only 0.03% of its protein mass present as particles. However, this number increased progressively to 0.4% as protein was recovered from the reflectivity cell in “after reflectivity” sample. When the formed layer was subjected to rinsing steps, the BW1 and BW2 samples showed significantly increased percentages (0.6% and 3.34%, respectively) of the protein mass that was recovered from the surface was present in aggregates/particles. For the samples other than a stock, a reported percentage is a fraction of protein per unit volume in the aliquot, that is, normalized based on concentration in the aliquot determined by UV spectrometry. The MFI results were categorized by grouping the detected particles as small (1–10 :m) and larger (10–100 :m) species. In all cases tested, the small particles (ECD below 10 :m) constituted over 90% of the overall count. However, in all three fractions (“after reflectivity” and “BW1 and BW2”), the modest shift in the particle count percentage toward larger particle

species ultimately resulted in a dramatic increase in the mass percentage of the large species, because of their much larger size. The IgG1 stock solution showed only 33% of the aggregated protein mass was because of the large particles, whereas in the “BW2,” this value reached over 99% (Fig. 7a). Changes in particle morphology were also monitored as this was also evaluable via MFI.11,30 Aspect ratio and ECD of the formed particles were chosen as parameters for description of particle shape/morphology, and were combined into 2-D histograms. Figures 7b–7d demonstrate the progression of the particle distributions and morphology for particles formed in the low-salt IgG1 solutions from small spherical aggregates (stock) to larger, anisotropic species that were obtained during desorption/rinsing steps. One unexpected observation is the difference in the dimensions of the particles in the stock solution and in “after reflectivity” solutions. The MFI profiles for Figure 7b and 7c show that particles increase in ECD, but also have more elongated shapes after the solution has contacted the SiOx surface. As the experiment proceeds through the desorption stage, the formed particles further increase their ECD and shape asymmetry. The elongated nature of the particles may be even more pronounced than reported here because of the limited sensitivity of MFI to particles smaller than 5 :m. In Figure 7, “BW1” is similar to the “after reflectivity” sample; however, BW2 demonstrates a detectable presence of large, approximately 40 :m particles at a high aspect ratio, suggesting the presence of fibrillar aggregates at this stage of the experiment. The visual examination of MFI images further supported an occurrence of long, twisted particles (Fig. 7f). Comparison with Higher Ionic Strength Conditions Stock solutions of IgG1 at pH 4.5 with 100 mM of added NaCl had elevated particle counts, compared with buffer-only conditions, even for solutions that had not been exposed to the SiOx surface (see Table 1). Overall, a 10-fold greater percentage of IgG1 was incorporated into micron-sized particles in the highsalt solution and 94% of all aggregated protein mass consisted of particulate species with a size above 10 :m. For antibody solutions that had contacted the SiOx surface (“after reflectivity” sample), this yielded a lower (6.5-fold) increase in the aggregated mass of protein when compared with 13.3-fold increase under the low-salt conditions. The initial step of desorption also resulted in a diminished amount of protein recovered and a markedly lower percentage of protein mass incorporated into the aggregates (Fig. 8a). On the order of 1% of the total protein obtained during the first buffer wash was incorporated into particulates. Only a subsequent buffer rinsing step induced a larger recovery of aggregated IgG1—reaching approximately 4% of protein included in particles for BW2. Finally, Figures 8b and 8c demonstrate that changes occurred in the size/shape distributions of particles in the BW2 sample compared with the stock solution of IgG1, for the pH 4.5 conditions with 100 mM added NaCl. The substantial widening of the aspect ratio distribution over the entire ECD range suggests the development of longer, anisotropic species in the second buffer rinse aliquot. Staining the different samples with ThT dye resulted in a significant positive ThT signal observed only for the BW2 solution (Fig. 8d). Illustrative MFI images from the particles observed in BW2, as shown in the insert of Figure 8d, further suggest that largest particles are reasonably fibrillar, with amyloid-like or non-native structural

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Figure 7. Microflow imaging results for IgG1 at pH 4.5, 30 mM citrate. (a) Normalized total percent of protein incorporated into particles between 1–10 :m (red) and 10–50 :m (blue) each aliquot type (see main text for additional details). (b–d) Contour plots of two-dimensional histograms for subvisible particle size (ECD) and morphology (aspect ratio) in solution for: (b) stock solution of IgG1; (c) solution collected after reflectivity; (d) solution after BW2, insert: illustrative images of larger aggregates observed in “BW2” sample, as determined by MFI. The absolute count of the particles is specified by the number on a given contour line, as well as by the color code (blue: highest particle counts.)

characteristics. As with the IgG1 pH 6.2 low-salt samples, the amount of the soluble aggregate was insufficient to be characterized by the available techniques.

gregate formation were observed in all cases. Small aggregates were identified by solution scattering techniques, SANS, and larger aggregates through particle counting using MFI. Tertiary structural elements of the aggregates were characterized through extrinsic fluorescence.

DISCUSSION An objective of the current work is to help understand how surface interactions can affect the aggregation propensity of proteins. Most investigations to date have focused on how solution properties influence adsorption of protein to various materials.40 A smaller number of studies have attempted to characterize the structure of these protein layers upon adsorption, but have not focused on desorption events or how details of the structure, packing, and interactions of proteins with the surface and with each other impacts the formation of net irreversible aggregates. NR was used here to determine the surface coverage and infer qualitative and, in some case, semiquantitative details of the spatial arrangement or structure of protein films formed on a solid SiOx–water interface, and how they change after simple buffer rinses. Correlations between changes in the protein layer and the propensity for agDOI 10.1002/jps.24429

Conformational Deformation on the Surface Correlates with Solution Aggregation Neutron reflectivity results at pH 3.5 suggest that aCgn forms a film representing effectively a monolayer of aCgn molecules. However, comparison of the 1-D profile of the surface-adsorbed protein to a profile calculated from the X-ray protein struc˚ wider than the nature showed a dimension roughly 10 A tive structure, suggesting a partially unfolded conformation (Fig. 3a). A large percentage, approximately 70%, of the accumulated protein was rinsed off of the SiOx surface by pure buffer. The desorbed protein consisted of both monomeric and soluble aggregate species, as indicated by SANS spectra (Fig. 4a). These aggregates were under the size resolution limit of the MFI instrument and were therefore considered to be less than approximately 1 :m in diameter.

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In contrast, the aCgn films formed at pH 4.5 and resolved by NR resulted in a profile that closely resembled the X-ray structure, suggesting minimal conformational changes for the adsorbed protein. The subsequent buffer rinse completely removed the protein layer (Fig. 3b) and no aggregates were found in the collected soluble fraction, either by SANS or MFI (Fig. 4b). The solution unfolding free energy for aCgn increases significantly by moving from pH 3.5 to 4.5. These results suggest that intrinsic protein stability may play a role in surface-adsorbed structure and subsequent aggregate formation. Modulating the unfolding free energy, by raising the solution pH, appears to have helped to retain the folded structure of aCgn on the surface and presumably then mitigated detectable aggregate formation. IgG Layer Structure

Figure 8. Microflow imaging results for IgG1 at pH 4.5, 30 mM citrate and 100 mM added NaCl. (a) Normalized total percent of protein incorporated into particles between 1–10 :m (red) and 10–50 :m (blue) each aliquot type (see main text for additional details). (b and c) Histograms for subvisible particle morphology (aspect ratio) in solution for: (b) stock solution of IgG1; (c) BW2 solution.

Immunoglobulin gamma orientation at the interface has been investigated previously.16,41–43 In a majority of studies, a predominantly flat-on conformation of immunoglobulin molecules was observed at both hydrophilic and hydrophobic surfaces. For example, Xu et al.15 demonstrated that a mouse anti-$-hCG antibody adopts a mostly flat-on orientation on hydrophilic silica. However, some groups have suggested that flat-on conformation of antibody switches to end-on when protein concentration in the solution is increased from 0.002 to 0.01 mg/mL or from 0.01 to 1 mg/mL.17,44 In addition, at least one study observed the formation of protein multilayers that was dependent on the initial solution concentration.15 Neutron reflectivity is sensitive to the ensemble averaged dimensions of proteins adsorbed on a surface. The results of IgG1 bound to a hydrophilic SiOx surface were interpreted here based on predominantly two populations of protein upon adsorption, a primary layer tightly bound to the SiOx interface and a diffuse overlayer that was easily removed through buffer rinse steps. A flat-on orientation best described the 1-D scattering length density profiles of the primary IgG1 layer derived from the reflectivity measurements. IgG1 adsorption over a 60-fold concentration range did not show a transition in protein orientation from the initial flat-on position. At pH 6.2, however, a broadening of the antibody depth profile was observed. Under low-salt conditions, the broadened profile allowed for several interpretations, including either a population of mixed orientations or a flat-on orientation with some protein domains protruding away from the surface. Further discrimination between these models using NR would require specific labeling of the antibody domains. At high salt, the broadening was much less pronounced, presumably indicating orientations that were in between flat-on and side-on orientations (Fig. 1). In addition to tightly bound monomolecular layer of IgG1 on the SiOx surface, small amounts of diffuse protein layer(s) were observed at both pH values (Figs. 5 and 6). Multilayers were also observed in previous studies of IgG adsorption.17,44 In previous studies, the volume fraction of a protein “overlayer” was dependent on IgG1 concentration in solution during the adsorption step. If the attractions between the solvent-exposed amino acids and the SiOx surface are relatively nonspecific—for example, based on attractions between the negatively charged SiOx and the (net) positively charged protein surface—then, it would be expected that a flat-on orientation would provide lower energy states that would be more difficult to desorb via wash steps or agitation.

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IgG Surface Coverage as a Function of Salt and pH It is evident from the present study that an interplay of forces governs the adsorption and desorption properties of monoclonal antibodies to hydrophilic SiOx surfaces. Electrostatics is often presumed to play a dominant role in these interactions because of the charged nature of SiOx–water interfaces, and in the case of the IgG1 studied here the protein takes on markedly different charge values at pH values of 4.5 and 6.2. The experimentally determined charge on the IgG1 shows an almost threefold increase in positive charge when lowering the pH from 5.5 to 4.5 (from +5.9 to +15.5), with the difference expected to be even more pronounced between the pH conditions chosen for the current work.34 The results here suggest that, at the pH values furthest from its pI, the IgG1 undergoes a strong and almost irreversible association with the SiOx surface. As Figure 5a indicates, at pH 4.5, the area fraction of the adsorbed protein layer remains unchanged after buffer rinse, with almost all desorption coming from the diffuse overlayer. At pH 6.2, the protein is expected to have approximately threefold lower net surface charge, and (as might be expected) the primary protein layer is significantly reduced by the buffer rinse step with a 75% decrease in the maximum area fraction (Fig. 5b). Despite the lower charge and therefore diminished surface affinity, however, the high pH sample had a 40% greater coverage on the SiOx surface compared with the pH 4.5 sample. The surface association of this IgG1 is driven by the competition of the electrostatic intermolecular repulsion between the proteins and the attraction between the SiOx–surface and the antibody. Previously, protein–protein interactions for this IgG molecule were shown to be highly sensitive to pH and ionic strength of the aqueous buffer. Measurements of second virial coefficient (B22 )30 showed that the addition of 50 mM sodium chloride to the solution at pH 4.5 shifted B22 from being slightly positive to largely negative. The change of sign of the second virial coefficient signifies a conversion from repulsive to attractive interactions, with repulsions arising from electrostatic interactions. Increasing the ionic strength of the solution is expected to simultaneously modulate protein–surface and protein–protein interactions. Adsorption of antibody in 100 mM NaCl aqueous buffer resulted in an overall decrease in total protein volume fraction at the surface at both pH values evaluated. However, the reduction in total protein bound was more pronounced at pH 6.2. In addition, the monolayer formed at pH 4.5 remained tightly bound at 100 mM NaCl and showed minimum desorption compared with the pH 6.2 condition. These results point to net protein charge and solvent ionic strength directly impacting the strength of protein–surface interactions. However, a greater protein surface coverage was still found at pH 6.2 than at pH 4.5, suggesting that protein– protein interactions while on the surface also influence the total fraction bound. Scenarios of either electrostatic surface–protein attraction or lateral protein–protein repulsion dominating protein adsorption events have been reported in the literature,20,40,45,46 and the outcomes appear to depend upon the specific charge distribution on the biomolecules. The complex effect of protein–protein and protein–surface interactions on adsorption/desorption properties of IgG1 are observed in the current results as well. Although the current experiments show “proof of principle” that both of these interactions can be modulated by pH and DOI 10.1002/jps.24429

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buffer ionic strength, a more systematic study would be required to map out the quantitative relative contributions of protein–protein and protein–surface interactions under different conditions.

Correlation Between Surface Association and Protein Aggregation The results of this work indicate that the interaction of proteins with hydrophilic SiOx surfaces can contribute to the formation of protein aggregates in the solution. The link between surface adsorption and aggregate formation was readily apparent in the case of aCgn, where strong surface interactions led to the formation of a dense protein film with the protein structure that is perturbed from the native fold. Santore et al.47 have previously observed the slight denaturing of fibrinogen on a hydrophilic surface by the use of an adsorption probe method, whereas Su et al.48 also observed a similar denaturation phenomenon for bovine serum albumin. However, lysozyme remained unperturbed on SiOx surfaces under a range of pH and ionic strength conditions,20 suggesting that stability at hydrophilic and hydrophobic interfaces can be a protein-specific property. The conformation of the structured domains of immunoglobulins associated with SiOx is not easily deduced by NR. Flexible linker regions join these structured domains and prohibit a detailed comparison with high-resolution structural data of the full-length IgG molecule. Although the overall dimensions of the experimentally derived NR profile are in general agreement with calculated values, perturbations to the native folding of individual domains or along the plane of the surface are not likely to be discernable by NR. Nonetheless, the subsequent analysis found a strong correlation between IgG desorption from SiOx and an increased number of particulates observed in solution by MFI. Transient exposure of the IgG1 studied here to the SiOx surface was sufficient to increase the population of micron-sized particles compared with monomeric protein. Figures 6a and 8a show that, for the protein solution collected immediately after flowing through the reflectivity cell (the “AR” sample), there was a 10-fold increase in the mass ratio of particles– monomeric protein, as compared with the stock solution, under both low- and high-salt conditions. This outcome may have been because of shear stress experienced by the sample during the flow step; however, this possibility seems unlikely in light of the very slow application of the material through the flow cell (shear rate = 300 s−1 ). The Reynolds number for the buffer rinse steps was much less than 1 (calculation not shown), indicating essentially creeping flow and low shear stresses at the liquid– solid interface. The most extensive formation of particles was observed in the “BW2” samples, in which the desorbed species can presumably only have originated from the primary IgG layer, as the BW1 step already removed residual solution in the cell, as well as the easily desorbed protein from the surface. The particles obtained throughout this phase were found to be fibrillar in nature based on particle size and aspect ratio (Figs. 6d and 8c). ThT fluorescence helped to demonstrate that these particles were significantly non-native and possibly amyloid-like in their structure. Strong binding of the ThT fluorescence probe was observed for particles in the BW2 sample for low- and highionic strength solutions (Supp Figs. 3A and 3B).

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The preceding buffer rinse step, “BW1,” resulted in the collection of the diffuse overlayer. In this case, particles were structurally similar to the “after reflectivity” samples. Interestingly, the amount of protein incorporated into the particles was significantly lower for a recovered IgG1 from BW1 then from BW2 for both the low- and high-ionic strength solutions, suggesting that protein adsorbed directly to the SiOx surface, although desorbed in smaller quantities, nucleates into large fibrillar aggregates in solution.

CONCLUSIONS Adsorption of proteins at various interfaces presents a potential risk during storage and manufacturing of biotherapeutics. The results here provide insights into the nature of protein– surface interactions, which can potentially aid in developing optimized molecular design, formulation, and production strategies to mitigate risks associated with surface–protein interactions. The amount of protein adsorbed to hydrophilic surfaces was quantified by NR, which provided both the thickness of the protein layer and the surface coverage. Compared with bulk solution concentration, only a small amount of protein comprises the surface-bound fraction and forms a welldefined monomolecular layer. Minimal amounts of stress (e.g., low-shear rates) were able to cause at least partial desorption. However, IgGs formulated away from their pI values, and therefore higher in their overall charge, bound more tightly to the SiOx, keeping the surface bound layer mostly intact and resulting in a lower yield of desorbed aggregates. Nonetheless, our studies show that in all cases exposure to the SiOx surface does accelerate particle aggregate formation, as a parallel pathway to thermally induced aggregation of IgG1 molecules. The nature of the surface-bound protein phase is proteinspecific, as demonstrated by "Cgn and IgG1, and is dependent on multiple characteristics, such as the surface-denaturating propensity of protein in question, colloidal stability, and structural flexibility. In addition to evaluating these protein characteristics, further work needs to be carried out to study these proteins at other manufacturing-relevant surfaces, such as plastics, metals, and oils. This type of information can boost an effort of molecular chemistry, manufacturing, and controls departments in designing the next generation of molecules meeting various manufacturing product quality challenges. Although it has been used for a number of years in other fields, NR is an emerging surface-sensitive technique from the perspective of biopharmaceutical development and product characterization, with the versatility to study a large array of surfaces with different chemical properties and to measure proteins without the need for labeling or other modifications. Working in a flow-cell environment allows for downstream (or upstream) characterization before exposure of proteins to the surface of interest. However, the requirement for neutron sources, which are limited to only several locations worldwide, does present limitations. For many applications, in-house X-ray scattering instruments may provide a viable alternative. In the past, X-ray reflectivity has been applied to study proteins and other biomolecules at the air–water interface and with careful sample cell development and the right X-ray target source, solid–liquid interfaces can also be measured. Therefore, further effort in developing a routine method for studying proteins adsorption/desorption under simulated manufacturing condition

is required and would benefit the larger biopharmaceutical science community.

ACKNOWLEDGMENTS T.P., H.N., and C.J.R. gratefully acknowledge GSK for generously supplying the IgG1 material, as well as the National Institute of Standards and Technology for providing the neutron facilities used in this work. This work was funded, in part, by support from the National Institutes of Health (R01 EB006006) and the National Institute of Standards and Technology (NIST 70NANB12H239).

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