Circular Dichroism Spectroscopy as a Tool for Monitoring Aggregation

Article pubs.acs.org/ac

Circular Dichroism Spectroscopy as a Tool for Monitoring Aggregation in Monoclonal Antibody Therapeutics Varsha Joshi, Tarun Shivach, Nitin Yadav, and Anurag S. Rathore* Department of Chemical Engineering, IIT Delhi, Hauz Khas, New Delhi 110016, India ABSTRACT: Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and farUV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate Tonset which has been found to correlate with aggregation induced by salt, low pH, and buffer species. Tonset temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

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tion.11 Because of the universal nature of protein aggregation and significant concerns related to its adverse effects, it is critical that the levels and sizes of aggregates be quantified accurately and reproducibly during processing, product characterization, and lot release. The extent of characterization has to be commensurate with the level of risk associated with each phase of drug development.15 Antibody aggregates can be characterized by a variety of analytical techniques.16−19 These include size exclusion-high performance liquid chromatography (SEC), analytical ultracentrifugation (AUC), field flow fractionation (FFF), dynamic light scattering (DLS), and mass spectrometry (MS). Further, tools such as Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), fluorescence microscopy, and fluorescence spectroscopy assess the state of folding/unfolding of the product and thereby can be used to monitor the structural change that might lead to aggregation. Each of these tools utilizes a different detection principle, and hence, industry

onoclonal antibodies (mAbs) represent the most rapidly growing product class of therapeutic protein.1−3 More than 25 therapeutic mAbs have been approved by the major regulatory agencies, and hundreds of others are currently in various stages of development.4 All of the approved therapeutic mAbs belong to the immunoglobulin G (IgG) class of molecules, and of them IgG1 is the most common immunoglobulin used for pharmaceutical and biomedical purposes. Despite the high quality of current therapeutic biotechnology products, protein immunogenicity remains a key concern. The presence of aggregates is considered an important product-related impurity that can increase the risk of immune responses.5,6 Hence, aggregates are considered as a critical quality attribute (CQA) for a therapeutic product.7−9 Antibody aggregation can occur throughout the manufacturing process at various steps including cell culture, harvest, purification, formulation, and filling, and during storage. Therefore, significant efforts are spent toward minimizing protein aggregation during these process steps.10−14 Monoclonal antibody therapeutics when exposed to changes in operating parameters such as pH, temperature, salt concentration, buffer composition, shear rate, and surfaces are prone to structural instability and protein−protein interactions, inducing aggrega© XXXX American Chemical Society

Received: July 1, 2014 Accepted: October 28, 2014

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dx.doi.org/10.1021/ac503140j | Anal. Chem. XXXX, XXX, XXX−XXX

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Table 1. Concentration of the Various Excipients in the Different Formulations Examined in This Studya

a

formulation

% tween 80

% trehalose

% sorbitol

monomer content (%) at 30 °C after 45 days

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

0.0003 0.0003 0.0003 0.0003 0.3 0.3 0.3 0.3 0.0003 0.3 0.1502 0.1502 0.1502 0.1502 0.1502

5 5 20 20 5 5 20 20 12.5 12.5 5 20 12.5 12.5 12.5

5 20 5 20 5 20 5 20 12.5 12.5 12.5 12.5 5 20 12.5

95.86 95.71 95.51 47.87 60.83 66.45 77.61 81.92 86.99 89.59 91.72 88.97 87.91 89.4 89.37

Base buffer used was pH 6.5, 25 mM sodium phosphate buffer, with 150 mM NaCl in all cases.

use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding aggregation. Next, thermal scanning CD has been used as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. Results from both studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

practice is to use a combination of these tools for understanding aggregation. SEC is the traditional workhorse for aggregation analysis of pharmaceutical proteins.20 It offers significant advantages over other analytical methods including high sample throughput due to autosampling capabilities, high resolution between the monomer and higher order oligomer species resulting in accurate quantification, and high precision. However, SEC analysis suffers from a few but significant limitations as well.17,21,22 First, interactions may occur in some cases between the aggregates and column matrix resulting in erroneous estimation of aggregate level. Second, dissociation or formation of protein aggregates may occur during the course of a sample run impacting accuracy. Third, very large aggregates (e.g., subvisible particles) may not be able to pass through frit and may not enter the column. It is for these reasons that it is industry practice to complement SEC data with data from other orthogonal analytical techniques. DLS is another tool that is used for characterization of aggregates. It is a nondestructive and easy to perform analytical technique with high sensitivity and low sample consumption. However, DLS too suffers from several disadvantages including complicated data analysis, lack of quantitation of aggregate level, and low resolution (weak differentiation between particle species).19 The sensitivity of DLS largely depends on the working environment and the precision of handling the protein sample without contaminants such as dust or foreign matter from the surroundings. With the sophistication in the instrumentation, the data analysis has become fairly robust, but one is dependent on algorithms from manufacturers. Insoluble aggregates can be removed by filtration, but soluble aggregates cannot be removed. Soluble aggregates also pose a threat due to associated immunogenicity problems.6 Protein aggregation is typically accompanied by significant changes in the secondary and tertiary structures of the protein.23 As aggregation can be correlated to changes in the secondary/ tertiary structure, CD has been used for monitoring conformational changes during protein aggregation. It offers a simple and reliable method for rapid determination of protein structure or for monitoring conformational changes.24 Thus, this paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the

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MATERIALS AND METHODS Immunoglobulin and Reagents. Monoclonal antibody IgG1 (pI 8.2−8.5) used in this study was provided by a biosimilar manufacturer. The antibody was stored at 4 °C at 37 mg/mL solution in 15 mM sodium phosphate with 150 mM NaCl at pH 7 in aliquots. The buffers used for the experiments were filtered with 0.22 μm nylon membrane filter and degassed. All chemicals and reagents were HPLC purity grade purchased from Sigma-Aldrich Co. LLC, India. Sample Preparation. Downstream Buffer Screening Experiments. Samples for the aggregation studies were prepared from IgG stock aliquots by performing buffer exchange using G-25 sepharose packed columns in respective buffers. Following buffer exchange, 3 mL samples were stored at chosen buffer conditions and temperature. Protein concentration was adjusted to 10 mg/mL, and the mAb sample was incubated at 4 and 30 °C. Analysis of samples was performed at 0, 6, 24, 120, and 168 h. Before analysis, the samples were centrifuged at 7000g for 5 min to remove insoluble aggregates. All the samples were examined for soluble aggregation using SEC and CD spectroscopy. Formulation Buffer Screening Experiments. Solutions were prepared using aliquots of IgG stock and by performing buffer exchange into the base buffer (25 mM sodium phosphate, 6.5 pH, 150 mM NaCl). The protein concentration was measured by UV spectroscopy at a wavelength of 280 nm and was adjusted to a concentration of 2.5 mg/mL. Thereafter, the excipients (polysorbate 80, D-trehalose, and sorbitol) listed in Table 1 were added to the sample as per the concentration listed. A 1 mL sample of each formulation was incubated at 4 °C as well as 30 °C, and analysis was done after 45 days. Before analysis, the samples were centrifuged at 7000g for 5 min to remove insoluble aggregates. All the samples were examined for B

dx.doi.org/10.1021/ac503140j | Anal. Chem. XXXX, XXX, XXX−XXX

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where μmax and μmin are the largest and smallest means, respectively, among the group of “a” sample means. MSE is mean square error, and n is the number of replicates. The q(a, f), is the upper percentage points of q, where f is the number of degrees of freedom associated with the MSE. For equal sample sizes, Tukey’s test declares two means significantly different if the absolute value of their sample differences (D-value) exceeds the value of Tα, which can be estimated as

soluble aggregation using SEC and for conformational changes using CD spectroscopy. UV Spectroscopy. The mAb concentration was measured by SpectraMax M2 (Molecular Devices, LLC, California) at a wavelength of 280 nm in a 96-well plate, assuming an extinction coefficient of 1.41 cm2/mg. The final protein concentration was obtained after subtracting the optical density of buffer solution (blank) and dividing by extinction coefficient. Size Exclusion Chromatography (SEC). Samples were analyzed by SEC using BioSuite 250, 5 μm, 7.8 mm × 300 mm column (Waters, Milford, MA) at 25 °C. Agilent 1200 Infinite series HPLC unit (Agilent Technologies, Palo Alto, California) was used and consisted of quaternary pump with degasser, an autosampler with cooling unit, and a UV detector. Each sample was eluted isocratically for 17 min at a constant flow rate of 0.8 mL/min. The mobile phase consisted of 100 mM phosphate, 100 mM Na2SO4, 0.05% NaN3 at pH 7, filtered with 0.22 μm nylon membrane, and degassed. Monomer and aggregates were quantified by calculating the percentage area under the respective peak in the SEC chromatograms using the Agilent Chemstation software. Elution peaks were detected via absorbance at 280 nm. If the peaks of monomer, aggregates, and fragments were not baseline resolved, a perpendicular was dropped in the valley between the different peaks at a fixed retention time of the peak to allow for quantitation. The mass recovery has been checked in SEC for downstream and formulation samples. The injection volume and the mAb concentration were kept constant for all the samples. The total area of the sample was monitored during the complete length of the experiment and was found to be constant throughout the period of analysis in case of high pH buffers. However, in the case of low pH buffers, some insoluble aggregates were generated toward the end of the incubation time. These were removed by centrifugation and filtration since the focus of this study was on monitoring the soluble aggregation. Far-UV Circular Dichroism (CD) Spectroscopy. Far-UV CD analysis was performed on a Jasco J-815 CD spectrometer (Mary’s Court, Easton, MD). The spectra were recorded over wavelength range 200−250 nm in a 1 mm cell at sample concentration of 0.2 mg/mL at 20 °C. The sample volume required for analysis was 0.4 mL. The final spectra were the average of 5 scans, and the analysis took about 5 min for all the scans. CD spectra of the buffer solutions in the appropriate cuvette were subtracted from the sample spectra and smoothed using the Savistky−Golay function before conversion to absolute CD values. The mean residual ellipticity (MRE) was calculated using a mean residual weight (MRW) of 113 for the antibody as per the following expression: [θ ]mrw, λ = MRW ×

θλ 10 × d × c

Tα = qα(a , f )

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RESULTS Conformational Analysis of mAbs Using CD Spectroscopy. CD spectroscopy is a frequently used technique for

(1) Figure 1. Comparison of far-UV CD spectra at 4 °C for mAb incubated for 6 h in 15 mM phosphate buffer at pH 6.5 (conformation intact, aggregate content of 4.3%) and for 6 h in 100 mM citrate buffer with 100 mM NaCl at pH 3 (conformation lost, aggregate content of 4.5%).

assessment of protein conformation in solution. Far-UV CD spectra (180−250 nm) are directly related to the secondary structure of the protein due to asymmetrical packing of the intrinsically achiral (planar) peptide bond groups.24

μmax − μmin MSE n

(3)

The MRE values at 218 nm for each time interval were chosen for calculation of mean, q, and Tα. The statistical analyses were performed to obtain significant difference between particular pair of spectra. The D-value serves the purpose of determining whether there is difference in two spectra in the same buffer set at different time points. Tukey's test should not be used to compare two different set of spectra (different base buffers). Thermal Unfolding by CD. Thermal stability of mAb samples was assessed at a concentration of 0.2 mg/mL by recording the spectra from 20 to 90 °C at 218 nm wavelength. Monoclonal antibodies have a predominant β-sheet conformation; hence, the spectra were recorded at 218 nm. A 1 mm path length cuvette was used for the thermal unfolding analysis. Temperature was increased at heating rate of 1 °C/min using the Peltier thermocouple and a time constant of 16 s. The total duration of the scan was 70 min. Signal from the buffer blank was subtracted from the sample scan, and the data was smoothed using the 10 point moving average calculated by Microsoft Excel (Microsoft Corporation, Redmond, WA). This allows us a more robust estimation of Tonset. The first derivatives of these smoothed spectra were able to show the exact Tonset temperature. This method of Tonset at 218 nm can be applied to mAbs or proteins with predominant β-sheet content.

Here, θλ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm), and c is the concentration (g/mL).25 Statistical Analysis of Spectra. CD spectra of the mAb in the DSP buffers were recorded in 5 replicates. Tukey’s test was performed to determine whether the means (average of 5 repeats) of the spectra differ significantly from each other and to the control sample at zero hour. The test statics was expressed as distribution of the studentized range statistic, q26 q=

MSE n

(2) C

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Table 2. Absolute Values of the D-Value (Difference in Means) of the Spectra at 218 nm and amongst Different Time Pointsa 4 °C, Ac3* buffer, Tα = 0.4

30 °C, Ac3* buffer, Tα = 0.5

time points (h)

D-value

change in % agg

D-value

change in % agg

0 and 6 6 and 24 24 and 120 120 and 168

1.1 0.44 0.78 0.15

1.22 0.00 9.00 0.13

1.92 0.72 0.69 0.6

47.07 18.32 11.63 9.80

Thus, CD spectroscopy can be effectively used for characterization of the secondary structure of a protein as well as for monitoring any conformational changes. The major advantage that CD spectroscopy offers over other analytical tools such as near-infrared (NIR) spectroscopy is that the spectroscopic signal is not affected by the presence of the buffer environment. Well-defined procedures based on reference spectra of different structural elements are available for elucidation of the secondary structure (e.g., K2D, CONTIN, LINCOMB, SELCON3, etc.).25 However, it is difficult to quantify minimum change that CD can detect and correlate it to aggregate content. Tukey’s test was performed to determine whether the means at different time points are same or different. The result of Tukey’s test for mAb in acetate buffer at different time points and the trend of change in aggregate content in that buffer set are shown in Table 2. It is seen that Tukey’s-test-based analysis of CD spectroscopy data can be used only to indicate if aggregation is expected or not and not to quantify the level of aggregation. In the following sections we present two applications of CD spectroscopy toward measurement of aggregate levels and examining stability of a protein therapeutic. Correlation of Far-UV CD with SEC. The mAb samples incubated in pH 3 acetate and glycine buffers were analyzed by far-UV CD spectroscopy for changes in their conformation (Figure 2A). Same samples were also analyzed by SEC for measuring aggregate levels (Figure 2B). The percentage of aggregates formed using buffers with salt showed a rapid increase with time (Figure 2B). This correlates well with the results of CD analysis (Figure 2A) which exhibit a significant increase in the depth and breadth of the far-UV CD spectra at 218 nm over time. The D-value for pH 3 acetate buffer with NaCl at 30 °C between 0 and 6 h is 1.92 which is +1.42 compared to the Tα (0.5) (Table 2). The data suggests

a

If D-value > Tα, the change is significant, or if D-value < Tα the change is insignificant. Ac3* = 100 mM pH 3 acetate buffer with 100 mM NaCl. The change in aggregate content (change in % agg) can be correlated for a single buffer at different time points. It should not be correlated among two different sets of spectra. The D-value serves the purpose of determining whether there is difference in two spectra in the same buffer set at different time points.

Different domains of the IgG molecule form compact globular structures with a characteristic fold. The predominant secondary structure elements in IgGs are the antiparallel βsheet and random coil conformations.27 Further, short stretches of α-helices (that are found in some bends) and β-turns are also present. IgGs largely consist of β-sheet structures (Fab = 47%, F(ab′)2 = 50−60%), whereas the α-helix content is 2−7%.28 The four primary classes of secondary structures for proteins, namely α helix, β sheet, β turn, and random coil, each exhibit absorption in a specific wavelength range.27 A broad minimum at 218 nm in the far-UV CD spectra is indicative of a significant presence of β-sheets. Changes in pH, temperature, and incubation time can cause alterations in the conformation. For example, the IgG used in this study retained its β sheet conformation (no change in spectra) in phosphate buffer at pH 6.5 at 6 h, but its conformation was lost in citrate buffer at pH 3 with 100 mM NaCl when incubated for 6 h at 4 °C (Figure 1).

Figure 2. Comparison of far-UV CD (A) to SEC (B) showing correlation between both the techniques for monitoring of aggregates of monoclonal antibody therapeutics. (A, B) CD spectra and the HMW (%) of mAb in 100 mM Glycine pH 3 with NaCl and 100 mM acetate pH 3 with NaCl at the intervals of 0, 6, 24, 120 and 168 h at 30 °C, respectively. D

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Figure 3. Comparison of thermal scanning CD (Left column A and B) and SEC (Right column A and B) for buffers: Ac6, 25 mM acetate pH 6; Ac6*, 25 mM acetate pH 6 with 200 mM NaCl; Ac3, 100 mM acetate pH 3; Ac3*, 100 mM acetate pH 3 with 100 mM NaCl; Ci6, 20 mM citrate pH 6; Ci6*, 20 mM citrate pH 6 with 200 mM NaCl; Ci3, 100 mM citrate pH 3; Ci3* 100 mM citrate pH 3 with 100 mM NaCl. The HMW (%) at 120 and 30 °C is chosen for comparison. (C) The first derivative spectra of the data from part A are shown with exact values of Tonset presented as a table.

conditions examined here. The increase in D-values with respect to time signifies increased change in the means of spectra among different time points. The D-value also correlates with trend of aggregate content between time points (Table 2). Similar results were obtained with pH 3 glycine with NaCl where the percentage of aggregates formed showed a rapid increase with time (Figure 2B). This also correlates well with the results of CD analysis (Figure 2A) which exhibit an increase in the depth and broadening of the far-UV CD spectra at 218 nm over time. Correlation of Thermal Scanning Using CD with SEC. Due to the transient perturbations in the molecular structure, heating may induce aggregation at conditions well below the melting temperature of the protein. Furthermore, increase in temperature accelerates aggregation due to its impact on the individual rate constants of aggregation kinetics. This is the reason why high temperature is often used to examine the stability of a protein.29 Spectroscopy has been shown to be an appropriate approach because it provides information about the protein structure at a submolecular level.23 As stated above, mAb products are predominantly β-sheet proteins and exhibit a decrease in the MRE values at 218 nm. Hence, to examine their thermal stability, transitions in the CD

Figure 4. Graph showing correlation between Tonset temperature and HMW (%). The low aggregate content samples showing high Tonset temperature (68−80 °C) and high aggregate content samples showing low Tonset temperature (