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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: J Pharm Sci. 2014 Jan 22;103(3):796–809. doi: 10.1002/jps.23839

Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

Srivalli N Telikepalli 1, Ozan S Kumru 1, Cavan Kalonia 1, Reza Esfandiary 1,2, Sangeeta B Joshi 1, C Russell Middaugh 1, David B Volkin 1,*
PMCID: PMC4040128  NIHMSID: NIHMS577804  PMID: 24452866

Abstract

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions.

Keywords: protein, aggregation, particles, formulation, monoclonal antibody, IgG, accelerated stability

INTRODUCTION

A current concern with the use of monoclonal antibody-based therapeutics is their tendency to aggregate and form particles during long-term storage and/or during accidental exposure to environmental stresses. The formation of aggregates and particles may lead to an increase in immune response 1-3 or a decrease in efficacy of the drug.2,4 Protein aggregation can occur during many stages of production (purification, formulation, and filling), or during long-term storage, shipping, and even administration to the patient.5 Therefore it is important to better understand the reasons for aggregation and particle formation due to different stresses and formulation conditions in order to develop strategies to minimize its occurrence.

Aggregation and particle formation in therapeutic protein formulations can be caused by a variety of environmental stresses or by formulation conditions such as concentration,6-8 solution pH,6,9,10 and the presence or absence of certain excipients.6,7,9 Freezing can not only lead to changes in the formulation pH6,11,12 and concentration of proteins and excipients,6 but also to the formation of ice/water interfaces6,13-15 where protein adsorption can induce partial protein unfolding and subsequent aggregation.6,9,15-17 Proteins subjected to heating undergo conformational changes that can lead to the formation of aggregates and particles.9,18 Mechanical stresses may cause shear or interfacial effects in which the protein adsorbs to the air-water interface, leading to structural alterations which can initiate aggregation as well.9,16,19,20 Stirring and shaking are both mechanical stresses that can also cause cavitation, local thermal effects, bubble entrapment, and transportation of the aggregated protein from the air-water or air-container interface into the bulk solution.6,21,22,23

One major challenge in studying protein aggregation experimentally is that a wide variety of analytical techniques are required to characterize the formation of protein aggregates and particles over a broad size range (from few nanometer to hundreds of microns).24 In addition, it is also important to have complimentary, orthogonal techniques for analyzing aggregates of similar size ranges since results can differ based on the principles and setup of each technique.6 In this work, we follow the previously proposed definitions of protein aggregates, across the size ranges of few nanometers to 100s of microns. 25 In this case study, size exclusion chromatography (SEC) is used to analyze smaller aggregates in the size range of tens of nanometers. Although it is a powerful analytical tool for monitoring small nanometer-sized soluble aggregates, upon injection of sample into the column, aggregates can potentially dissociate upon mixing with mobile phase or adhere to the column thereby requiring careful method development and use of orthogonal techniques.26 For sizing submicron particles (0.1 to 1 μm), Nanosight Tracking Analysis (NTA) is used while for micron (1-100 μm) size particles, Microflow-Imaging technique (MFI) is employed. NTA tracks and sizes individual particles (unlike its DLS counterpart), but has limited sensitivity in detecting low numbers of submicron particles. In addition to sizing and counting particles like light obscuration, MFI also has digital imaging capabilities that can provide morphological information allowing differentiation between silicone and protein particles. To detect visible particles larger than 100 μm, visual assessments are employed. Turbidity is also used as a general method to monitor the formation of aggregates and particles in solution across the various size ranges. Although visual inspection is a commonly used technique, even under pre-defined conditions and with extensive analyst training, results may vary between different analysts. Turbidity provides semi-quantitative information for comparisons of the overall aggregation state of a sample, but it does not provide information regarding the size or number of particles. Detailed discussions of the strengths and limitations of these analytical techniques are described more thoroughly elsewhere.27,28

Recently, there has been an increased emphasis on characterizing the morphology and composition of particles in addition to counting and sizing them.27 As a starting point for this work, MFI analysis, in addition to sizing and counting of subvisible particles, is used for morphological analysis by utilizing parameters such as aspect ratio and intensity of the digital images.29,30 For visualization of small, nanometer-sized aggregates, transmission electron microscopy (TEM) is used.31 SDS-PAGE is used to determine the extent of covalent, disulfide linkages present in aggregates.32 Extrinsic fluorescence spectroscopy with 8-anilino-1-naphthalene sulfonate (ANS) probe provides information concerning the surface hydrophobicity of aggregates.33;34,35 FTIR provides insights into the secondary structure of native protein and aggregates in solution,6,36-38 with the use of a FTIR microscope allowing for selection of individual protein particles for secondary structure analysis.29

This paper is a “protein particle formation” case study by utilizing a variety of analytical techniques to examine the effect of four different environmental stresses (freeze-thaw, shaking, stirring, and heating) and formulation composition (salt concentration) on the number, size range morphology, and compositional nature of the IgG1 aggregates/particles formed from stressing the mAb solutions. Additionally, a new data visualization method consisting of radar plot analysis was used to better evaluate the effects of environmental stress and salt concentration on particle size distributions as well as changes in certain morphological parameters measured by MFI. The trends observed in terms of types and amounts of particles formed under the different stress conditions are discussed, along with some comparisons to previous studies with different monoclonal antibodies.

EXPERIMENTAL METHODS

Materials

Purified monoclonal human IgG1 (mAb) was obtained from Janssen Biotech (Radnor, PA) at 40 mg/mL. The reagents and stir bars required for sample preparation were purchased from Sigma Aldrich (St. Louis, MO), and Fisher Scientific (Pittsburgh, PA). The 3 mL vials and rubber stoppers used to generate protein aggregates were purchased from West Pharmaceuticals (Lionville, PA). For counting and sizing of aggregates (using SEC, NTA, MFI, turbidity, visual assessments), the mAb was diluted to 1 mg/mL using 10 mM sodium acetate buffer, pH 5 ± 150 mM NaCl.23 This condition was also used for morphological analysis of particles by MFI. For structural and morphological analysis of the aggregates (using TEM, MFI, SDS-PAGE, FTIR-Microscopy, ANS-Fluorescence), the mAb was diluted to 1 mg/ml in 10 mM sodium acetate buffer, pH 5 containing 150 mM NaCl.

Generation of Aggregates

The 40 mg/mL IgG1 mAb solution was diluted to 1 mg/mL in 10 mM sodium acetate buffer, pH 5 ± 150 mM NaCl and then subjected to a variety of accelerated stress conditions. These conditions were selected to match pH solution conditions used previously with a different set of IgG mAbs23,39 In each case, the buffer controls were stressed similarly to the protein samples and analyzed using various sizing, counting, and characterization techniques. Since the buffer controls showed very low particle counts, the data were not included in the figures. For freeze-thaw stress, the mAb was frozen and thawed one to three times (indicated as cycles) at -80°C and room temperature, respectively (labeled FT-C1 and FT-C3). For shaking stress, the mAb was agitated at 300 rpm (using an IKA AS260.1 shaking platform) for 1-3 days (labeled shake-D1 and shake-D3). For stirring stress, the mAb was stirred at an intermediate speed (setting 5) on a stirring plate (ThermoSci Pierce Reacti-Therm III #18823 Heating/Stirring Module) using Flea Micro Teflon coated magnets (Fisher Scientific) for 1-3 days (labeled stir-D1 and stir-D3). For thermal stress, the mAb was incubated at 60°C in an incubator (Revco Ultima II) for 1-3 days (labeled heat-D1 and heat-D3).

Size-Exclusion Chromatography

A Shimadzu Prominence HPLC system equipped with a diode-array detector was employed with a Tosoh Bioscience TSK-Gel Bioassist G3SWXL (7.8 mm × 30.0 cm) PEEK column and a corresponding PEEK Guard column (TSK Guard Column SWXL, 6.0 mm × 4.0 cm) that were preconditioned with BSA as described previously.26 Molecular weight standards (Biorad Laboratories; Hercules, CA) were run to test for efficiency of separation and resolution. Both the column and guard column were equilibrated at 30°C for 1 hour using the mobile phase comprised of 0.2 M sodium phosphate, pH 6.8 at a flow rate of 0.7 ml/min. Aggregated samples were centrifuged at 16,000g (851 rotor on an IEC Micromax 3593) for 5 min and 10 μL of supernatant was injected for analysis and monitored simultaneously at 214 and 280 nm for each 30 min sample run.26 Multimers, dimers, monomer, and fragment peaks were quantified using the LC Solutions data analysis software provided with the instrument as described elsewhere.26

Nanoparticle Tracking Analysis (NTA)

Submicron (nanometer) sized particles were measured using a Nanosight LM-14 (Nanosight, Amesbury, UK) with a high resolution EMCCD camera. Stressed mAb solutions were centrifuged at 16,000g for 5 min and 300 uL of the supernatants were injected into the sample holder. The stirred (with and without NaCl) and heat stressed (with NaCl only) samples were diluted by a factor of 100 prior to analysis. Three 30 s movies were taken at ambient temperature for each sample at a viscosity of 0.95 cP. Data analysis was completed using NTA 2.3 software (Nanosight) with the required camera level and gain adjustments. Dilution factors were accounted for in the data analysis.

Micro-Flow Digital Imaging

Subvisible (micron sized) protein particles were analyzed and imaged using a MFI DPA 4200 (Protein Simple, Santa Clara, CA). Prior to each analysis, the instrument was primed with purified water to obtain a particle-free baseline. Samples were gently swirled and 1 mL of each sample was removed using a low protein-binding pipette tip and loaded into a sample holder. Some of the samples (stir-D1 and D3 with and without NaCl; shake-D3 with NaCl; and heat-D3 with NaCl) were diluted by a factor of 100 prior to being passed through the instrument at a flow rate of 0.1m/min. The data were obtained as described previously by Kumru et al.40

Data Visualization with Radar Plots

To generate MFI particle size distribution radar plots, subvisible particle concentrations and sizes for both unstressed and stressed samples were obtained from MFI’s MVAS 1.3 software. Similarly, MFI particle morphology radar charts were created using the average mean intensity and aspect ratio values for each sample. All samples were run independently three times (n=3). The data were pre-processed in Excel and two radar plots (one showing average values and one showing variability in the runs) were generated using the MiddaughSuite software created in our lab.41 The two radar charts were superimposed using Adobe Photoshop CS6. See Kalonia et al. 2013 and Kim et al. 2012 for a more detailed description of this data visualization methodology as applied to protein aggregation and conformational stability data, respectively.41,42

SDS-PAGE

Samples were centrifuged at 16,000g for 5 min to separate the soluble fraction (supernatant) from the insoluble fraction (pellet). Both fractions were dissolved in NuPAGE LDS sample buffer (Life Technologies, Carlsbad, CA) with and without 50 mM DTT (BioRad) and incubated at 80°C for 90 s. Approximately 10 μg of each sample was separated on a 3-8% Tris-Acetate gel using Tris-Acetate running buffer (Life Technologies) for 65 min at 150V. A Hi-Mark unstained molecular weight ladder was used as a reference (Life Technologies). The starting protein concentration for the supernatant of one sample (stir-D3) was low so a maximum of 20 μL was loaded. Protein bands were visualized by staining with Bio-Safe Coomassie blue (BioRad).

Turbidity

A HACH 2100 AN turbidimeter was used to monitor the turbidity of each of the samples. Prior to running the aggregated samples, NTU calibration standards were used to generate a standard curve.

Transmission Electron Microscopy (TEM)

Carbon-coated grids were dipped into methylene chloride for 10 s to remove the top carbon layer and were dried for a few minutes. The images were obtained by uranyl acetate staining and by following the procedure by Kumru et al.40

Free Thiol Quantitation

Samples were centrifuged at 16,000g for 5 min to separate the soluble (supernatant) and the insoluble (pellet) fractions. The pellet was dissolved in 6M guanidine hydrochloride (Fisher Scientific). The amount of free thiol in the samples as well as in the appropriate controls was measured using the protocol described in the Measure-iT Thiol Assay Kit (Molecular Probes) with a SpectraMax MS plate reader (Molecular Devices; Sunnyvale, CA).

Extrinsic Fluorescence Spectroscopy

Samples were centrifuged at 16,000g for 5 min to separate the soluble and insoluble fractions. The pellet was resuspended in 10 mM sodium acetate, 150mM NaCl, pH 5. The protein concentration of each of these supernatant and pellet components was measured using a Nanodrop spectrometer (Thermo Scientific) with light scattering correction. The samples were diluted to 0.1 mg/mL in 10 mM sodium acetate, 150 mM NaCl, pH 5. 8-Anilinonaphthalene-1-sulfonate (ANS; Sigma-Aldrich, St. Louis, MO) was added and the ANS fluorescence of the samples were recorded according to Kumru et al.40 The signal from the buffer with equivalent amount of ANS was subtracted from all measurements.

Fourier Transform Infrared Microscopy

Five μm gold filters (Pall Corporation) were used for analyzing the aggregated samples. Filters were equilibrated by washing with 0.1 M NaOH. The samples were then filtered, washed with ultrapure water, and dried overnight. A Bruker Hyperion FTIR Microscope with a 15X objective was used to image individual particles. Two-hundred-fifty-six scans were recorded from 600-4000 cm-1 with a viewing area of about 100 μm × 100 μm. To observe the maximum change in secondary structure due to heating, a 1 mg/mL sample in acetate buffer (10 mM sodium acetate, 150 mM NaCl, pH 5) was heated for 20 min at 80°C. This heated sample (labeled heated control) was also filtered onto the 5 μm gold filter and dried overnight. OPUS (V6.5) software was used for baseline and atmospheric correction. The second derivative spectra were obtained using a nine-point Savitzky-Golay smoothing function.

Fourier Transform Infrared Spectroscopy

The unstressed mAb at 1 and 10 mg/mL in 10 mM sodium acetate, 150 mM NaCl, pH 5 were analyzed with a Bruker Tensor 27 FTIR Spectrometer and a Bio-ATR cell. Two-hundred-fifty-six scans were recorded from 600-4000 cm-1 at a resolution of 4 cm-1. To observe changes in secondary structure as a function of temperature, 256 scans of 10 mg/mL of unstressed sample were heated from 10-87.5°C at 4 cm-1 resolution and 120 s equilibration time with 2.5°C increments.

RESULTS

Counting and Sizing of Aggregates and Particles Formed under Accelerated Stress Conditions

SEC (soluble aggregates, <100 nm)

To determine the amount of smaller (soluble) nanometer aggregates, the stressed IgG1 mAb samples were centrifuged and the resulting supernatants were analyzed by SEC. The amount of protein material that did not elute from the SEC (referred to as insoluble aggregates) was indirectly determined by monitoring the decrease in the total area of the chromatogram peaks between unstressed (D0) and the stressed samples. The earliest eluted peak was labeled as multimer, the second peak as dimer, the third and largest peak as monomer, and the final peak as fragment based on the estimated molecular weights. In the absence of salt, heat stressed samples produced more fragments, dimers, and insoluble aggregates compared to the unstressed sample (Fig. 1A, I). Stirring formed more multimers and insoluble aggregates (Fig. 1G), while freeze-thaw formed a very small amount of multimers and dimers (Fig. 1A), compared to the unstressed sample. Shaking (Fig. 1E) did not show an increase in any species relative to the unstressed sample. In the presence of 0.15M NaCl, protein was further destabilized and generally resulted in the formation of more aggregates. Heat-stressed mAb in the presence of NaCl (Fig. 1J) showed more insoluble aggregates, a larger decrease in monomer, and an increase in multimer compared to its NaCl-free counterpart, which showed some fragment formation and lower levels of impurity (Fig. 1I). Stirring the mAb solution in the presence of salt resulted in a large increase in insoluble aggregate and a concurrent decrease in monomer content (Fig. 1G vs. H). Freeze-thaw and shaking did not show changes in any species, but in the presence of NaCl, a slight increase in insoluble aggregate was observed (Fig 1D, 1F).

Figure 1.

Figure 1

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Formation of soluble and insoluble aggregates in IgG1 mAb solutions exposed to different stresses as measured by SEC. (A, B) Representative SEC chromatograms monitored at 214 nm of 1 mg/mL antibody in 10 mM sodium acetate, pH 5 in absence (A) and presence (B) of 150 mM NaCl. Key: Day 0 Control (—), F/T Cycle 3 ( Inline graphic), Agitation Day 3 ( Inline graphic), Stirring Day 3 ( Inline graphic), Heating Day 3 ( Inline graphic). Plots of monomer loss and changes in amounts of impurities as a function of different stresses are shown: freeze-thaw cycles (C) without NaCl and (D) with NaCl; days of agitation (E) without NaCl and (F) with NaCl; days of stirring (G) without NaCl and (H) with NaCl; days of heating (I) without NaCl and (J) with NaCl. Key for C-J: Inline graphic -% Monomer; Impurity:▬- % Insoluble; ▲- % Multimer; ▼ - % Dimer; ◆- % Fragment. Each graph represents the average of three separate experiments (n=3). Error bars represent the 95% confidence interval.

Nanoparticle Tracking Analysis (NTA) (50-1000 nm particles)

NTA was used to assess the concentration and size distribution of nanometer sized particles (also referred to as submicron particles) formed in stressed and unstressed samples. Stirring of the mAb solution in the absence of NaCl (Fig. 2A and 2C) generated the largest number of nanometer sized particles (between ~150-250 nm). In contrast, in the presence of salt, heating generated the most nanometer sized particles (Fig. 2B and 2D). The concentration of nanometer particles present in the unstressed controls was below the instrument’s quantitation limit (data not shown).

Figure 2.

Figure 2

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Formation of submicron sized particles in IgG1 mAb solutions exposed to different stresses as measured by NTA. Representative NTA data showing the formation of nanometer sized particles due to four indicated stresses applied to 1 mg/mL antibody solution containing 10 mM sodium acetate, pH 5 in the (A) absence of NaCl and (B) in the presence of NaCl. Concentration of nanometer sized protein particles formed due to each stress at day 1 (D1) and (D3) in the presence and absence of NaCl (C, D). Each data point are the average of three separate experiments (n=3) and the error bars for each data point represent the 95% confidence interval.

MFI (2-100 μm particles)

Subvisible particle data (concentration and size range of micron size particles) obtained from MFI measurements of stressed and unstressed samples were visualized using radar plots (Fig. 3 and Supplemental Fig. S1). This novel data visualization method for MFI data is described in detail elsewhere.42 As shown in the key to Figure 3, the concentric circles represent concentration of particles, with the lowest concentration (102) being the innermost circle, and highest concentration (>107) being the outermost. The 5 corners of the polygon within the circles represent the particle size bins. An increase in size of the polygon towards one corner represents an increase in concentration of particles in that size bin (e.g., stretching of a corner from the innermost concentric circle to the outermost circle in size bin number 3 indicates that there is an increase in the ≥10<25 μm particles from 102 to >107 particles/mL).

Figure 3.

Figure 3

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Radar plots for visualizing formation of subvisible particles (concentration and size distributions) in IgG1 mAb solutions exposed to different stresses as measured by MFI. Radar plots show MFI particle concentration and size data distributions as generated by four indicated stresses when applied to 1 mg/mL antibody solution containing 10 mM sodium acetate, pH 5 with and without 150 mM NaCl. See text for details of radar plot analysis. The data shown are the average of three separate experiments (n=3) and the error represents the 95% confidence interval.

As shown in Figure 3, the size and concentration of subvisible particles formed is highly dependent on the type of stress. In the absence of NaCl, freeze-thaw and heat stressed mAb samples showed the lowest concentration of particles even after 3 cycles/days of stress, while shaking and stirring showed a higher concentration of particles. Stirring produced the most subvisible particles, especially in the 2-25 μm size range. In the presence of NaCl, a higher concentration of particles, especially in the 2-25 μm size range, was observed in all of the stressed mAb samples. The heated mAb showed a large difference in both particle number and size in the presence and absence of salt that increased from day 1 to day 3. A large variability in the larger particles was observed (the lighter shading on the radar plots reflects the standard deviation of the measurements). In general, shaking the mAb solution containing NaCl generated the largest subvisible particles (2-50 μm) (Fig. 3). Particle counts of controls were negligible.

Turbidity and Visual Assessment

Results of turbidity measurements and visual observations of the stressed mAb solutions are shown in Table 1. In samples lacking NaCl, agitation and stirring resulted in the highest number of >100 μm (referred to as visible particles, VP), with stirring producing the greatest turbidity. No major NaCl effect was seen for the freeze-thaw stress. Agitation, stirring, and heating stresses on the mAb formed more VP and higher turbidity in the presence of NaCl than in the absence of it. The highest turbidity levels were measured for stir-D3 samples, followed by stir-D1, and heat-D3. In addition, the presence of NaCl increased the turbidity for the heated (heat-D1 and heat-D3) protein compared to the corresponding heat stressed mAb samples without NaCl. Shaken or stirred mAb samples formed a large number of visible particles regardless of the presence or absence of NaCl.

Table 1.

Turbidity measurements and visual assessments of visible particles were performed on control and stressed mAb samples. Samples contained 1 mg/mL antibody in 10 mM sodium acetate, pH 5 in absence and presence of 150 mM NaCl. The turbidity data are an average of three independent experiments (n=3) with standard deviation. For visual assessments of visible particles, “-“ is no change observed relative to unstressed sample; “+” some change observed relative to unstressed sample (2-5 particles detected); “++” notable change observed relative to unstressed sample (> 5 particles detected).

Turbidity (NTU) Visual Assessment Turbidity (NTU) Visual Assessment
No NaCl With 150 mM NaCl
mAb Samples Average Stdev Average Stdev
Buffer Alone (no mAb) 0.2 0.1 - 0.7 0.6 -
Day 0 0.6 0.1 - 0.7 0.1 -
Freeze-Thaw Cycle 1 1.3 0.3 - 0.7 0.1 -
Freeze-Thaw Cycle 3 1.5 0.5 - 1.6 0.2 -
Agitation Day 1 8.1 1.6 ++ 22.4 2.1 ++
Agitation Day 3 7.6 0.4 ++ 25 10.5 ++
Stirring Day1 13.2 5.4 ++ 217.7 17 ++
Stirring Day3 64.4 7 ++ 756.7 71.9 ++
Heating Day1 0.9 0.1 - 82.4 3.4 +
Heating Day3 0.9 0.1 - 212.3 5.9 ++
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Structural Characterization of Aggregates and Particles

Particle Morphology

As an initial step in characterizing the nature and composition of the particles formed under different stresses, MFI was used to elucidate morphological information (intensity and aspect ratio parameters) of the subvisible particles generated after three days of stress (Fig. 3 and Supplemental Fig. S1). Aspect ratio is a ratio of width of the particle relative to the height and intensity is related to the particle absorption characteristics. Data were visualized in the form of a radar plot (Fig. 4). Figure 4 shows the different stresses applied to mAb solutions with and without NaCl on the x-axis and the MFI morphology parameters, intensity and aspect ratio, on the y-axis. The top and bottom radar charts describe the intensity and aspect ratio, respectively, of the particles as a function of stress and solution condition. As shown in the key to Figure 4, the aspect ratio of the micron particles varies from ~0.35 (elongated) to 0.85 (more circular) and the intensity from about 350 (opaque) to 850 (highly transparent) intensity level units (ILU). It should be emphasized that the concentric circles here do not represent concentration, but rather a change in particle morphology with the outermost circle representing elongated, opaque particles, and the innermost circle representing transparent, circular particles. The corners of the 5-sided polygon represent distinct particle size bins (labeled 1-5 in the figure). The pink-shaded region indicates that an insufficient number of particles were collected in that size range for accurate morphological analysis.

Figure 4.

Figure 4

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Radar plots for visualizing morphology parameters of subvisible particles (aspect ratio and intensity) in IgG1 mAb solutions exposed to different stresses as measured by MFI. Radar plots show MFI morphology data distributions as generated by four indicated stresses when applied to 1 mg/mL antibody solution containing 10 mM sodium acetate, pH 5 with and without 150 mM NaCl. See text for details of radar plot analysis. The data shown are the average of three separate experiments (n=3) and the error represents the 95% confidence interval.

In the absence of NaCl, the shaking of the mAb solution over 3 days resulted in particles that appeared transparent (Fig. 4A) and elongated (Fig. 4B) over all particle size ranges. In the presence of NaCl under the same stress condition, the particles formed are less transparent (i.e., more opaque) and less elongated (i.e., more circular in shape) over a narrower size range. Stirring-induced mAb particles formed in the presence or absence of NaCl do not show notable differences in intensity or aspect ratio. The concentration of particles in the freeze-thaw and heated samples without NaCl was lower (Fig. 3) and an accurate comparison of the morphology change across the size ranges was limited (as shown by the shaded areas in Figure 4).

To characterize the morphology of smaller particles, TEM was used to examine the shape of nanometer sized aggregates generated from 3 days of different stresses in the presence of 0.15 M NaCl. Some representative TEM images are shown in Figure 5. It can be seen that the type of stress to which the mAb solution was subjected influences the morphology of the aggregates generated. Freeze-thaw and heated aggregates appear very fibrillar, while agitated-induced aggregates appear fibrillar with some spherical aggregates. The stirring-induced aggregates were predominantly spherical in nature in this size range.

Figure 5.

Figure 5

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Representative TEM images of IgG1 mAb aggregates and particles formed after 3 freeze-thaw cycles or three days of each indicated stress. Particles were isolated from 1 mg/mL antibody solution in 10 mM sodium acetate, 150 mM NaCl, pH 5.

Non-native disulphide cross-linking

SDS-PAGE was used to study the formation of non-native covalent crosslinking in the stressed mAb. Results for the stirring and heating stressed mAb containing NaCl are shown in Figure 6A and B (non-reduced and reduced, respectively). These samples were first centrifuged to separate the supernatant (S) and pellet components (P) prior to electrophoresis. The non-reduced supernatants contained predominantly monomers while the pellets contained high molecular weight species above 500 kDa (i.e., larger than tetramer). Upon reduction of the supernatant and pellet, a complete loss in the high molecular weight material was observed with the light and heavy chains bands of the mAb visible as the primary species. The pellet for stir-D1 and D3, heat-D1 and D3 contained significant amounts of non-native disulfide linked aggregates. The supernatant of heat-D1 protein contained some of these non-native disulfide linked aggregates as well. No disulfide-linked aggregates were detected in freeze-thaw and agitation stressed mAb samples (data not shown). The number of free sulfhydryl groups in the mAb samples was also measured, and virtually no free thiols were present in control or stressed samples (data not shown).

Figure 6.

Figure 6

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Reduced and non-reduced SDS-PAGE gels of IgG1 mAb samples exposed to four different stresses. Samples contained 1 mg/ml antibody in 10 mM sodium acetate, 150 mM NaCl, pH 5. High molecular weight aggregates formed by disulfide linkages were observed in pellet of stir-Day 1, stir-Day 3, heat-Day 1 and heat-Day 3 samples. Stressed samples were centrifuged to separate supernatant (S) from pellet (P), run on SDS-PAGE, and stained as described in methods section.

Overall secondary structure content

The secondary structure of the aggregated samples was studied using FTIR spectroscopy of control mAb samples and FTIR microscopy of individual particles formed by stress in 10 mM acetate, 150 mM NaCl, pH 5. Representative spectra are shown in Figure 7 along with wavelength values from multiple measurements including standard deviations. Two mAb controls were prepared for comparison to the particles formed from stressed samples: an unstressed mAb with native conformation (Figure 7A in the form of second derivative FTIR spectra) and particles isolated from an extensively heated mAb sample with some degree of structural perturbations (Figure 7B). Figures C-F show microscopic images and the corresponding FTIR spectra of isolated amorphous mAb particles generated under different stresses.

Figure 7.

Figure 7

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FTIR analysis of overall secondary structure of IgG mAb solutions and isolated particles as generated from four indicated stresses. Samples contained 1 mg/ml antibody in 10 mM sodium acetate, 150 mM NaCl, pH 5. Second derivative FTIR spectrum of (A) native, unstressed protein in solution, and (B) mAb heated at 80°C for 20 min to determine the maximum extent of secondary structure loss in solution, (C, D, E, F) Representative optical images of isolated mAb particles (by passing through gold filter) as generated from the four indicated stresses and their corresponding second derivative FTIR spectra from FTIR microscope. Numerical values are the average of three separate experiments (n=3) with error associated with the wavenumbers representing the 95% confidence interval.

In the Amide I region of the second derivative FTIR spectra of the native mAb, two minima at 1637 cm-1 and 1690 cm-1 were observed (Figure 7A). This result is consistent with the predominantly intramolecular beta sheets present in native IgGs. 9,43,44 For the isolated mAb particles from the extensively heat stressed control, severely altered secondary structure displaying extensive loss of intramolecular beta sheet structure (as shown by loss of the two minima seen in the native mAb control, and the appearance of intermolecular beta sheets with minima at 1617 cm-1, and 1693 cm-1 (Figure 7B) is seen. For the isolated mAb particles produced by three days of heating at 60°C, the spectra show the sample is more similar to the extensively heated control as evidenced by the presence of the two minima at 1624 cm-1 and 1694 cm-1.45,46 The mAb particles isolated after three freeze-thaw cycles and three days of agitation have FTIR spectra with primarily native-like structure as seen by the second derivative minima at 1634 cm-1 and 1691 cm-1 and 1635 cm-1 and 1692 cm-1, respectively. The stirred aggregates show reproducibly shifted minima in the main second derivative peak to 1631 cm-1 indicating some alteration in secondary structure.

Surface hydrophobicity

To examine any changes in the exposure of apolar regions in the stressed samples containing NaCl due to structural perturbations or aggregation/self-association, ANS extrinsic fluorescence spectroscopy was used. Stress induced aggregated mAb samples were centrifuged and separated into supernatant and pellet components. They were analyzed along with two controls: the unstressed mAb (D0) and a positive control (Heat-melt). As a positive control to determine the greatest extent of apolar binding of ANS, the mAb solution was heated from 10 to 87.5°C in the presence of ANS. From the emission spectrum, it was determined that the maximum binding of ANS to the mAb occurred at 72°C suggesting maximal ANS access to apolar sites at high temperature. Representative spectra are shown in Figure 8. The control mAb solution (D0) along with supernatants from the FT, shake, and stir-D1 samples (Fig. 8A,B) show very little fluorescence intensity indicating that in these samples, ANS has very little access to the apolar regions of the mAb. In the stir-D1 pellet, slightly higher fluorescence intensity was observed, which suggests some exposure of apolar sites in these aggregates (Fig. 8A, B). The heat-D1 supernatant and pellet show the largest ANS fluorescence intensity increase suggesting the greatest exposure of apolar regions. The heat stressed mAb samples containing NaCl displayed a similar magnitude of fluorescence intensity as the positive control (heat-melt) indicating that this stress condition may also generate samples with extensively apolar exposed regions.

Figure 8.

Figure 8

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ANS extrinsic fluorescence analysis of IgG1 mAb samples (supernatant and pellet components) before and after indicated stress was applied to samples. Samples contained 1 mg/ml mAb in 10 mM sodium acetate, 150 mM NaCl, pH 5. (A) Representative ANS spectra, and (B) ANS fluorescence intensity values (at 480 nm) for each stressed mAb sample compared to the two controls: unstressed (D0) and extensively heated samples (Heat-melt). The average intensities shown are based on three separate experiments (n=3). The error bars represent 95% confidence interval.

DISCUSSION

As an initial set of experiments, we first counted and sized protein particles generated under accelerated stress conditions employing an IgG1 mAb solution with and without 0.15 M NaCl. A summary of the counting and sizing results from SEC, NTA, MFI, Turbidity, and Visual Assessments are presented in Table 2. Different stresses (in the presence and absence of NaCl) result in the formation of mAb aggregates and particles of varying sizes which cannot be measured by a single analytical method. As shown in Table 2, using multiple techniques is therefore very important for analyzing the formation of a broad size range of aggregates. For example, SEC showed that the formation of soluble aggregates was influenced by the presence of NaCl across the four stresses. NTA results demonstrated that the presence of NaCl affected the extent of nanometer particle formation in the freeze-thaw and heat stressed samples, but not for the agitated and stirred stressed samples. Similarly, MFI detected that NaCl enhanced the formation of micron particles only for the freeze-thaw stressed mAb sample. Finally, turbidity and visual assessments showed changes in particle formation (due to the presence or absence of NaCl) with heat stressed mAb solutions.

Table 2.

Summary of formation of soluble aggregates and nanometer, subvisible (microns), and visible (>100μm) sized particles as measured by SEC, NTA, MFI, Turbidity, and Visual Assessments, respectively. Symbols describe whether a change was observed (+) or not observed (-) compared to the unstressed mAb sample with the given analytical technique. Eight types of measurements are shown in the table and the last column summarizes, for each stress, the number of notable effects (+ vs. -) due to the presence vs. absence of NaCl observed for each assay. Samples contained 1 mg/mL antibody in 10 mM sodium acetate, pH 5 in absence and presence of 150 mM NaCl.

SECa NTA MFI Turbidity Visual Assessment # Results differed (±NaCl)
%M %I %A %F
Freeze/Thaw
 -NaCl - - - - - - - -
 +NaCl - + - - + + - - 3/8
Agitation
 -NaCl - - - - - + + +
 +NaCl - + - - - + + + 1/8
Stirring
 -NaCl - + - - + + + +
 +NaCl + + - - + + + + 1/8
Heating
 -NaCl - + - + - + - -
 +NaCl + + + - + + + + 6/8
Open in a new tab
a

%M, %I, %A, %F correspond to percent monomer, insoluble aggregate, soluble aggregate, and fragment, respectively.

Freeze-thaw stress was observed to be the mildest condition in solutions without NaCl since essentially no detectable aggregates or particles were seen (Table 2, first row). Upon addition of NaCl (Table 2, second row), some insoluble aggregates (detected as a loss of total mass by SEC) and some nanometer and micron particles were detected by NTA and MFI. Shaking the mAb solution with and without NaCl (third and fourth row in Table 2) generated a turbid solution with primarily micron (2 to >100 μm) size particles observed by MFI and visual inspection. The shaken solution in presence of NaCl was more turbid with some insoluble aggregates, and a larger number of micron-sized particles 2 to >100 μm. Stirring without NaCl produced a moderately turbid solution consisting of some insoluble aggregates, submicron, micron, as well as a large number of visible entities. Stirring the mAb solutions, in the presence of NaCl, generated the most turbid solutions consisting of a very large amount of insoluble aggregates (20% after day 1 and around 60% after 3 days of stress, as quantified by SE-HPLC). The largest differences in aggregate and particle formation were observed due to heating the mAb solution (Table 2; rows seven and eight). Heating in the absence of NaCl generated a fairly clear solution consisting of primarily monomers with a very small amount of insoluble aggregates and fragments (SEC), little to no nanometer-sized (NTA), micron (MFI), and visible particles even after three days of stress. In the presence of NaCl, however, a highly turbid solution formed containing much more insoluble and multimer aggregates as well as a large increase in the number of nanometer, micron, and visible sized particles.

Comparison of these results to other studies shows similar trends in which mAb aggregate and particle formation depends on the type of stress and solution conditions. For an IgG1 mAb with a basic pI range formulated at lower solution pH such as pH 5.0, the addition of NaCl (<0.15 M) may promote protein aggregation and particle formation during environmental stress by a combination of effects resulting from neutralizing protein surface charge and decreasing electrostatic interactions. These effects may promote protein-protein interactions (colloidal stability) or decrease the structural integrity and stability of the protein (conformational stability).9,47 The last column of Table 2 describes the impact of NaCl on various stresses using eight analytical readouts obtained from four instruments. For a given stress, column values can potentially range from 0/8, indicating NaCl had no impact on the aggregation behavior, to 8/8 where NaCl impacted the aggregation behavior of the mAb as measured by all of these analytical methods. Agitation and stirring stresses of the mAb solution were least influenced by the presence of NaCl (1 out of 8). Although the two stresses themselves were damaging to the protein, the addition of NaCl minimally increased mAb instability (as manifested in detectable changes in SEC for percent insoluble aggregate and monomer content for agitation and stirring stresses, respectively). Aggregation of the mAb under freeze-thaw stress was more influenced by the presence of NaCl (3 out of 8). Although freeze-thaw stress does not seem to cause alterations in the conformation of the mAb (see below), increases in percent insoluble and soluble aggregate, as well as nanometer and micron sized particles (by SEC, NTA, and MFI, respectively) were noted due to the presence of NaCl, indicating decreased colloidal stability. Finally, heat stress showed the largest effect of NaCl on mAb instability (6 out of 8). While heating of the mAb solution in the absence of salt alters the conformational stability of the protein, it does not result in extensive aggregate or particle formation. With the addition of NaCl, however, there is now a large colloidal component to the mAb instability.

Despite the availability of new analytical methods to accurately and precisely count and size protein particles in the submicron and subvisible size range,27,28,48-51 biophysical characterization of the nature, composition and structural integrity of the protein within these particles remains a major analytical challenge. The focus of this work was to characterize aggregates and particles formed in an IgG1 mAb in the presence of 0.15M NaCl as a function of various stresses (freeze-thaw, shaking, stirring and heating). The primary goal was to characterize the morphology, structural integrity and composition of the IgG1 particles formed in 0.15M NaCl containing solutions by probing the extent of non-native covalent cross-linking, changes in overall protein secondary structure, and alterations in surface hydrophobicity within the particles as formed by each type of stress applied to the mAb solutions. Additionally, we also evaluated the morphology of these particles by TEM and by a newly developed approach from our laboratory to analyze MFI size distribution and morphological data using radar plots.42 These plots are commonly used to summarize large sets of protein data from multiple biophysical techniques. Radar plots are used to better visualize particle size distribution and morphological parameters of the particles.42

The freeze-thaw stress appeared to be mild and essentially did not change the overall higher order structure or covalent crosslinking of the protein within the aggregates. Particles, isolated onto a gold filter, showed no detectable alterations in overall secondary structure relative to the native IgG sample, even after three freeze thaw cycles. The nanometer sized aggregates appeared to have a fibrillar morphology by TEM. The micron sized particles (10-50μm) were opaque (less transparent) and more elongated. Similarly, freeze-thaw samples showed very low surface hydrophobicity almost equal to that of the native protein. There appeared to be no covalent cross-linking present in the sample and no corresponding higher molecular weight aggregates seen on SDS-PAGE gels (data not shown). Results obtained from similar experiments by Joubert et al. as well as Barnard et al and Zhang et al.52,53 with different mAbs were consistent with our observations.23

For mAb solutions stressed by shaking, a common stress encountered by many protein drugs during transportation, generated predominantly fibrillar and slightly spherical nanometer particles, while the micron sized particles were more opaque and circular than the samples shaken without NaCl, as shown in Figures 4 and 5. These stressed samples contained little to no covalently linked aggregates with largely native-like secondary structure and low surface hydrophobicity (Figs 6-8) even after three days of shaking stress. While shaking generated higher number of aggregates and particles than freeze-thaw, there were not many differences in the biophysical characteristics of the aggregates generated by these two stresses compared to the unstressed, native protein. This result is similar to data published previously with other proteins.17,23,54

Stirring stress was a harsher condition on the IgG1 compared to freeze-thaw and shaking. In addition, the particles generated from this stress were predominantly spherical in morphology which was not observed with other stresses. From the MFI morphology data, NaCl did not notably affect particle morphology (in terms of aspect ratio and intensity) of the stirred samples in the micron size range as shown by the radar chart analysis in Figure 4. Biophysical differences were also observed in protein from the pellet fraction and supernatant fraction of the centrifuged stirred IgG mAb samples. The protein in the pellet fraction displayed greater surface hydrophobicity (Figure 8) and contained an increased amount of reducible high molecular weight covalently linked aggregates (Figure 6) than the supernatant fraction. The ANS fluorescence data suggest that the population of aggregates present in the pellet may be more structurally altered compared to the aggregates present in the supernatant. The isolated stir-stressed particles showed some loss in overall intramolecular beta sheet structure content and some increase in the formation of intermolecular beta sheet structures (non-native, aggregated structures) as shown by FTIR analysis (Figure 7). One study reported that the overall secondary structure of the stirred sample was similar to that of the unstressed sample, contrary to our results with this IgG.17 In addition to rapid transportation of aggregates into bulk solution from the interface, protein also encounters the harsh shear force of the stir-bar and the resulting thermal effects as well as cavitation.17,55 All of these factors could account for higher aggregation and particulation with stirring stress compared to shaking.

For the heated samples of the IgG mAb, the supernatant fraction contained less non-native covalently linked aggregates compared to the pellet fraction which possessed higher levels of this type of disulfide cross-linked aggregate as measured by SDS-PAGE (Figure 6). The variability in the micron particle concentration was very high due to heating, so it is difficult to make definitive conclusions about the aspect ratio or intensity of these particles, although more elongated and more opaque particles were seen in the presence of NaCl (Figure 4). In the presence of NaCl, nanometer sized particles seen by TEM appeared fibrillar in morphology (Figure 5). The heat stressed samples displayed a highly perturbed overall secondary structure content consisting of predominantly intermolecular beta sheets as measured by FTIR (Figure 7). The increased ANS binding in both the supernatant and pellet of the centrifuged heated samples suggests significant exposure of apolar moieties in both fractions. Hawe et al. noted that the heat generated aggregates contained non-native covalent cross-linking with significantly increased ANS binding (increase in surface hydrophobicity), as well as a large perturbation in the secondary structure 18,23 Zhang et al. conducted a similar experiment with Bevacizumab and determined that heat generated aggregates had significantly altered structure from their ANS and intrinsic Trp fluorescence data.53 These results are consistent with our observations

In summary, while there has been a vast growth in our ability to count and size protein aggregates and particles over a wide size range during the past ~5 years, our analytical capabilities to describe the morphology, structural integrity and composition of the protein within these particles is still limited. The goal of this study was to provide a case study to examine the effect of different environmental stresses on an IgG1 mAb solution in terms of the morphology of particles formed as well the extent of structural alterations of the protein within the particles. We confirmed results of previous studies 17,18,23,52,55 that the type of stress a mAb solution experiences greatly influences many of its physical properties in distinct ways. There are still many gaps in our understanding that need to be addressed, 56 especially if there is a relationship between a particular particle count, particle size range, particle weight or perhaps the physicochemical or morphological trait(s) of protein particles/aggregates and their potential to generate an immune response in vivo. 56-62

Supplementary Material

Supp Figure 01

Supplemental Figure 1. Total concentration of subvisible particles (greater than 2 μm as measured by MFI) formed in IgG1 mAb solutions as generated by indicated stress at different time points. Samples contained 1 mg/ml antibody in 10 mM sodium acetate, pH 5 in the absence (A) or presence (B) of 150 mM NaCl. The MFI particle data shown are the average of three separate experiments (n=3) and the error bars represent the 95% confidence interval.

Acknowledgments

The authors wish to thank and acknowledge Janssen R&D for providing the IgG1 mAb for this study and the Kansas Bioscience Authority for financial support. ST financial support was provided by NIH biotechnology training grant 5-T32-GM008359. We would also like to thank Dr. Prem Thapa, Dr. David Moore, and Heather Shinogle of the KU Microscopy and Analytical Imaging Laboratory for helping us obtain TEM images.

Footnotes

SUPPORTING INFORMATION AVAILABLE

This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com.

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Associated Data

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Supplementary Materials

Supp Figure 01

Supplemental Figure 1. Total concentration of subvisible particles (greater than 2 μm as measured by MFI) formed in IgG1 mAb solutions as generated by indicated stress at different time points. Samples contained 1 mg/ml antibody in 10 mM sodium acetate, pH 5 in the absence (A) or presence (B) of 150 mM NaCl. The MFI particle data shown are the average of three separate experiments (n=3) and the error bars represent the 95% confidence interval.

NIHMS577804-supplement-Supp_Figure_01.tif (3.8MB, tif)

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