Purity Determination by Capillary Electrophoresis Sodium Hexadecyl

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Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel application For Therapeutic Protein Characterization Jeff Beckman, Yuanli Song, Yan Gu, Sergey Voronov, Naresh Chennamsetty, Stanley R. Krystek, Nesredin A. Mussa, and Zheng Jian Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03831 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Analytical Chemistry

Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CESHS): A Novel application For Therapeutic Protein Characterization

Jeff Beckman*1, Yuanli Song1, Yan Gu1, Sergey Voronov1, Naresh Chennamsetty2, Stanley Krystek3, Nesredin Mussa1, and Zheng Jian Li1.

1

Biologics Development, Bristol-Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA.

2

Biophysical Characterization Group, Bristol-Myers Squibb Company, 311 Pennington Rocky Hill Road, Pennington, NJ 3

Drug Discovery Research, Bristol-Myers Squibb Company, R. 206 and Province Line Road, Princeton, NJ

*Corresponding Author: Jeff Beckman, PhD

[email protected]

Phone: 978-784-6916 Fax: 978-784-6381

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ABSTRACT

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Capillary Gel Electrophoresis using sodium dodecyl sulfate (CE-SDS) is used commercially to provide

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quantitative purity data for therapeutic protein characterization and release. In CE-SDS, proteins are

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denatured under reducing or non-reducing conditions in the presence of SDS and electrophoretically

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separated by molecular weight and hydrodynamic radius through a sieving polymer matrix. Acceptable

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performance of this method would yield protein peaks that are baseline resolved and symmetrical.

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Nominal CE-SDS conditions and parameters are not optimal for all therapeutic proteins, specifically for

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Recombinant Therapeutic Protein-1 (RTP-1), where acceptable resolution and peak symmetry were not

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achieved. The application of longer alkyl chain detergents in the running buffer matrix substantially

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improved assay performance. Matrix running buffer containing sodium hexadecyl sulfate (SHS) in-

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creased peak resolution and plate count 3 and 8-fold respectively compared to a traditional SDS-based

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running gel matrix. At BMS we developed and qualified a viable method for the characterization and

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release of RTP-1 using an SHS-containing running buffer matrix. This work underscores the potential

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of detergents other than SDS to enhance the resolution and separation power of CE-based separation

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methods.

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INTRODUCTION

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Commercialization of Therapeutic Proteins require analytical techniques that can measure product

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heterogeneity from the complexity of biosynthesis (1,2). Towards this end, Capillary Gel Electrophore-

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sis (CGE) contributes to our understanding of protein size heterogeneity by separating and quantitative-

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ly detecting proteins by molecular weight and hydrodynamic radius (2-4). In this procedure, the protein

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is denatured with charged detergent to produce protein-detergent complexes with a uniform mass/charge

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ratio that is separated by molecular weight while sieving through a capillary filled with a hydrophilic gel

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buffer solution. This is followed by UV detection ideally at a point along the capillary when quantifica-

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tion of peaks can occur, which requires adequate protein peak separation efficiency (PSE), as defined by

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high plate counts and resolution. CGE works well for most therapeutic proteins in this regard, specifi-

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cally for IgGs, and has been accepted as standard for the evaluation of product purity in the biotechnol-

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ogy industry (5).

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Sodium dodecyl sulfate (SDS) has been used as the default detergent for CGE separations largely be-

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cause of its establishment in conventional polyacrylamide gel electrophoresis (SDS-PAGE) (6,7). In

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addition, SDS is able to uniformly bind to a typical protein at a ratio of 1.4 g SDS to 1 g protein, ensur-

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ing uniform mass/charge ratios of SDS:Protein complexes in most cases (8). Hence CGE is commonly

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referred to as SDS-CGE or CE-SDS.

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However, there are situations where CE-SDS yields poor protein PSE, which suggests relatively low

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SDS binding affinity and/or incomplete denaturation, which calls for an alternative method evaluation to

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obtain the optimal purity data.

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For this purpose we investigated alternatives to SDS. It is known that not all proteins bind SDS uni-

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formly due to the charge or glycosylation profile of a protein, e.g. (9,10), and numerous studies have

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demonstrated that the regulation of SDS affinity to protein requires specific modifications to the deter3 ACS Paragon Plus Environment


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gent and/or protein (10-14). For example, elongating the SDS hydrocarbon chain from 12 to 14 carbon

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lengths was shown to increase its protein affinity, underscoring the importance of hydrophobicity (14).

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Thus, for proteins that exhibit poor PSE by CE-SDS it is reasonable to postulate that an increase in de-

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tergent hydrophobicity can improve PSE by increasing the affinity of the detergent to the protein. Fig-

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ure 1 shows overlay electropherograms of RTP-1, an Fc-Adnectin fusion protein with a molecular

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weight of ~ 75 kDa (for a review of the characteristics of the Adnectin domain refer to (15)), run under

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typical CE-SDS conditions and with gel matrix containing sodium hexadecyl sulfate (SHS). The pres-

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ence of SHS led to a discernible improvement in PSE, resulting in baseline separation of the main Pro-

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tein peak from a prominent impurity peak (Impurity Peak 1, or IP1, in Figure 1) which was identified as

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a fragment of RTP-1 of ~ 2 kDa less than the parent molecule (data not shown). The improved peak

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shape and resolution extended the working range for the assay with the potential to increase assay sensi-

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tivity without compromising linearity. Specifically, the sample load could be increased 3-4 fold, im-

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proving signal-to-noise by a comparable magnitude, and allowing better quantification of low abundant

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peaks without causing significant migration overlap of the main and IP1 peaks (Figure 2; SHS Gel Buff-

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er data). Overlap in this case led to a decrease in the relative amount of IP1 which compromised the as-

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say by overestimating sample purity (Figure 2, SDS Gel Buffer data). Potential mechanisms for the im-

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provements observed are discussed.

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MATERIALS AND METHODS

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Reagents. Glycerol (≥ 99%), Ethylenediaminetetraacetic acid (EDTA, ≥ 99%) dextran (MW ~ 2000

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kDa), Tris(hydroxymethyl)aminomethane (Trizma, ≥ 99.9%), Boric acid (≥ 99.5%), and β-

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mercaptoethanol (≥ 99%) were purchased from Sigma (St. Louis, MO). Powders of sodium undecyl

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sulfate (SUS, ≥ 99%), sodium tetradecyl sulfate (STS, > 95%), and sodium hexadecyl sulfate (SHS, > 4 ACS Paragon Plus Environment

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98%) were purchased from Alfa Aesar (Wood Hill, MA). Powdered sodium dodecyl sulfate (SDS, >

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99%) was purchased from Avantor (Center Valley, PA). For CGE assay applications, 10 kDa internal

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standard (I.S.), 0.1 N acid/base wash solutions, SDS-MW gel and SDS-sample buffers, pre-assembled

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bare-fused silica capillary cartridges, and 2 mL universal vials and caps were purchased from AB Sciex

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(Framingham, MA).

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Sample Preparation. Unless stated otherwise, protein samples were prepared at 0.9 mg/mL with

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0.76% SDS, 5% β-mercaptoethanol and 76 mM Tris-HCl. 500 mM Tris-HCl pH 9.0 was prepared using

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a Schott pH meter equipped with an SI Analytics probe then deionized water and detergent powder add-

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ed to reach the desired concentrations. For relatively hydrophobic detergents, dissolving the powders

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into the Tris-HCl buffer required a combination of sonication and heating in a 70° water bath.

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Preparation of Gel Buffer Solutions. Gel buffers were prepared as follows: Trizma base, boric acid,

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EDTA, and glycerol were mixed together and filtered through a 0.2 micron filter. Detergent(s) and dex-

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tran were added subsequently. Once all components were in solution, the liquid was slowly poured into

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appropriately sized PTFE bottles purchased from Thermo Scientific (Waltham, MA) and stored at room

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temperature.

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Capillary Electrophoresis. CGE experiments were performed on a PA800+ instrument equipped

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with a photodiode array detector and 32Karat acquisition software (Version 9) (AB Sciex, Framingham,

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MA). Electrophoretic separations occurred in 50 µm internal diameter pre-cut capillaries at 15kV con-

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stant voltage with detection positioned 20 cm from the point of sample injection. Data from 32karat

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was transferred to Empower 3 (Build 3471, Waters, Milford, MA) for data processing. Note that Em-

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power is a chromatography-based software, thus migration times were converted to retention times and

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calculated as such when assessing PSE, see Equations (1) and (2) below.



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Assessment of Peak Separation Efficiency (PSE). PSE was evaluated using the following equations for plate count (Equation (1)) and resolution (Equation (2)) (United States Pharmacopeia, Chapter 621). Empower calculates these values automatically as field options. N = 16(Rt/W)2

(1)

Where N = the number of theoretical plates (plate count), Rt is the retention/migration time of the peak, and W the peak width at baseline with tangents drawn to 61% of peak height. R = 2(Rt2 – Rt1)/(W2 + W1)

(2)

Where R = the resolution between two peaks, Rt1 and Rt2 the retention/migration times of peaks 1 and 2 respectively, and W2 + W1 the sum of peak widths at baseline with tangent lines drawn at 50% peak height. Differential Scanning Calorimetry. RTP-1 was diluted with 1 x PBS (150 mM NaCl and 20 mM phosphate pH 7.2) to 0.5 mg/mL with various amounts of SDS or SHS. Measurements were performed on a Malvern MicroCal VP-DSC system (Malvern Instruments, Northampton, MA) with a cell volume of approximately 0.5 mL. Temperature scans were conducted from 20 to 95°C at a scan rate of 1°C/min. A buffer reference scan was subtracted from each sample scan prior to concentration normalization. Baselines were created in Origin 7.0 (Origin Lab, Northampton, MA) by cubic interpolation of the pre- and post-transition baselines. Protein Modeling. The amino acid sequences of one of the two domains (domain 2) of RTP-1 was aligned to a similar domain with known structure as the template (data not shown). The 3D structures of the domain were built using the homology modeling tool MODELLER (16). The homology models were subjected to side chain optimization and minimization steps and followed by model validation. The surface charge of the domain was calculated according to the charge of residues and the accessibil6 ACS Paragon Plus Environment

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ity of the residue in the Propka output file using Adaptive Poisson-Boltzmann Solver (17). Finally, the electrostatic map was visualized by the program PyMOL (Schrödinger, LLC).

RESULTS AND DISCUSSION To keep the focus primarily on detergent hydrophobicity and its impact on PSE, the detergents evaluated all contained the same charged sulfate head group and sodium counter-ion, only varying in alkyl chain length. These included sodium undecyl sulfate (SUS), SDS, sodium tetradecyl sulfate (STS), and SHS. These detergents have alkyl chain lengths of 11, 12, 14 and 16 carbons respectively. This series was chosen because detergents with carbon lengths less than 10 weakly bind to protein (18) and, in our experience, carbon lengths longer than 16 are not readily soluble in the gel solutions. To directly attribute alkyl chain length to potential improvements in RTP-1 PSE, initial experiments included only the detergent of interest in both the running gel buffer and sample solutions. As shown in Figure 3, PSE significantly improved with longer alkyl chain length, with SHS improving plate count over SDS by 8fold (11820/1450, Figure 4) and resolution between the main peak and the impurity IP1 by 2.3-fold (1.8/0.8, Figure 4). SUS marginally improved PSE compared to SDS, a phenomenon observed previously with the slightly smaller sodium decyl sulfate (18) and was attributed to the ability of detergents with smaller alkyl chains to more uniformly coat elongated proteins (albeit with lower affinity). Interestingly, these authors also found that STS and SHS reduced PSE of their model proteins by PAGE (18). (We also observed this phenomenon. Refer to the section below entitled “Impact of SHS on the CGE Performance of Other Proteins”.) One explanation is that RTP-1 is not represented by these model proteins, as may be the case given its properties (discussed below).



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Note that the CGE resolving power by longer chain detergents was not simply dependent on concentration since SDS could not achieve the same level of PSE as SHS regardless of concentration (Figure 4). The Correlation Between Longer Hydrocarbon Chain Detergents and the Improved Resolution of RTP-1. Hypothetically, more stable detergent:protein complexes should result in higher separation efficiencies because the structure would be more homogeneous (18). Based on specific physical properties of RTP-1, SDS may be insufficient to stabilize a uniformly denatured RTP-1 complex and instead may require a more hydrophobic detergent. The Adnectin domain of RTP-1 is thermophilic, a property that suggests its overall structure is rigid (19) and contains a relatively hydrophobic core (20). Under native or denaturing conditions (with or without SDS), thermophilic proteins are often required to overcome higher transition-state energy barriers towards denaturation compared to typical proteins (19,21). Modeling studies show that one face of the Adnectin domain of RTP-1 has a high proportion of negative charge potential which would cause electrostatic repulsion of a detergent sulfate group. This repulsion may require the more hydrophobic tail of SHS to establish an overall energetically-favorable interaction with the hydrophobic core of the protein to initiate unfolding (Figure 5). Differential Scanning Calorimetry (DSC) was used to quantify the energy requirements of SHS and SDS to fully denature RTP-1 (22). Endotherm profiles of detergent:RTP-1 complexes were biphasic, represented as peaks E1 and E2 in Figure 6. E1 and E2 are attributed to RTP-1 Fc and Adnectin domains respectively (data not shown). Thermodynamic data are summarized in Table 1. The observed enthalpy changes during thermal denaturation of the Fc domain, represented by the E1 peak, are comparable for both detergents (Figure 6C). However, the thermal denaturation profiles of the thermophilic Adnectin, or E2, were different depending on the detergent as shown in Figure 6D. Less energy was required for SHS to denature this domain, specifically in the range of expected critical micellar concentrations


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(CMCs), which would be less than 8 mM for SDS and less than 2 mM for SHS (23) and suggests that the SHS:RTP-1 complex is more stable with SHS bound. Impact of SHS on the CGE Performance of Other Proteins. The antibody (mAb) RTP-2 also required SHS to ensure adequate CGE performance (Figure 7A). In contrast, mAb RTP-3 yielded comparable CGE performance using SDS or SHS (Figure 7B). Note that SDS added to SHS gel buffer solutions improved the PSE of the 10 kDa protein internal standard without impacting the PSE of RTP-1 (I.S.) (Figure 8). For this reason, CGE performance was optimized using a gel containing a mixture of SDS and SHS. Predicting When a Protein Requires SHS in the Gel Buffer. Given that the hydrophobic property of SHS is likely responsible for its relative impact on PSE, we attempted to correlate increased CGE resolving power using SHS and protein hydrophobicity in order to predict when to incorporate SHS into the gel. Each protein was ranked by its Spatial Aggregation Propensity (SAP) score, which measures the proportion of hydrophobic patches on the protein surface (24). The correlation appeared weak, however, since both relatively hydrophilic and hydrophobic proteins required SHS to improve CGE performance (data not shown). Alternatively, it appears that proteins with unusually high thermal stability require SHS to optimize CGE performance. This prediction is based on the characteristics of the Adnectin domain, which is both highly thermostable and resistant to SDS denaturation (Figure 6 and (15)). RTP-2 fit this prediction since its highest transition Tm exceeded that of RTP-3 by ~ 10°C (data not shown). Overall, the intention of this work is to make available a second viable option if SDS does not deliver optimal resolving power. It would be ideal to establish a model that would predict when to use SHS. For this, more relevant Protein examples are needed that would cover wide ranges in thermal stability 9 ACS Paragon Plus Environment


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and other physical properties, such as molecular weight. The trend in the biotechnology industry is to engineer proteins to have relatively high thermal stability so as to improve pharmaceutical stability, e.g. to maximize shelf-life (25), thus we should encounter increasingly more proteins like RTP-1 and -2. Development of an SHS-Containing Gel Buffer and Qualification of a CE-SHS Purity Method for Product Release. The SHS gel buffer composition was developed to be robust, reproducible and stable, with an expiry of ≥15 months. The final RTP-1 purity method was successfully qualified with a nominal protein load 3-4 fold higher than otherwise obtained under fully optimized CE-SDS conditions (Figure 2).

CONCLUDING REMARKS SDS has long been the default detergent used for size-separation electrophoresis since the inception of PAGE (6), with some studies attempting to identify superior alternatives (18,26-28). Otzen reviewed recent efforts and concluded that SDS remains the best detergent for PAGE (7). This work underscores how alternative detergents can be of potential use to improve CGE resolving power of some proteins and related impurities. SHS is an example of one of these alternatives, with its effect likely induced by specific thermophilic/hydrophobic properties of select proteins. We postulate that similar improvements would be observed by SDS-PAGE using SHS with RTP-1 and RTP-2.

ACKNOWLEDGMENT The authors wish to thank Dr. Jeff Meyer and Devi Visone from Zymogenetics, a subsidiary of BristolMyers Squibb, Seattle, WA for supplying us with RTP-2 material and for helping to confirm the applicability of the SHS gel to RTP-2, and to Dr. Ming Zeng from Bristol-Myers Squibb, New Brunswick, 10 ACS Paragon Plus Environment

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NJ for participating in the initial assessment of the SHS gel.

We would also like to thank Drs. Julia

Ding and Roberto Rodriguez for their critical review of the manuscript.

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Manning, M., and Colon, W. Biochemistry 2004, 43, 11248-11254

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Banerji, A., and Ghosh, I. PLoS One 2009, 4, e7361

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Cunningham, E. L., Jaswal, S. S., Sohl, J. L., and Agard, D. A. Proc. Natl. Acad. Sci. USA 1999, 96, 11008-11014

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TABLES Table 1. RTP-1 Peak Separation Efficiency (PSE) Values Obtained Using Various Gel Buffer Detergents Detergent in the Gel Buff- Resolution Between Main Peak Theoretical er Solution (0.2%)a Main and IP1 Plates SUS (C11) 1.18 2530.0 SDS (C12) 0.77 1825.7 STS (C14) 1.63 6083.5 SHS (C16) 1.80 11815.9 a The RTP-1 sample solution contained the corresponding detergent


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Table 2. DSC functions obtained from adding various concentrations of either SDS or SHS to RTP-1 Detergent

SDS

SHS

a, b c

[Detergent], mM None

Tm1 (°C)a 68.9

Tm2 (°C) b 77.1

E1, ΔH1 (cal/mol)(105) c 3.20

E2, ΔH2 (cal/mol)(105) d 1.68

0.050

68.9

77.1

3.35

1.81

0.10

69.1

77.1

3.87

2.03

1.0

63.9

77.1

1.29

2.17

8.0

50.4

74.7

0.53

1.16

None

68.9

77.1

3.20

1.68

0.0030

68.8

77.0

3.20

1.88

0.013

66.7

77.1

2.01

2.18

0.50

60.6

77.2

0.45

1.17

2.0

58.7

72.4

0.24

0.87

Comparable drop in Tm with [detergent] regardless of detergent type

Comparable drop in ∆H1 with [detergent] regardless of detergent type (Figure 6C)

d

∆H2 dropped more dramatically with SHS after reaching a particular concentration (0.1 mM SDS and 0.013 mM SHS) (Figure 6D)


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FIGURES

Figure 1. CGE Electropherograms of RTP-1 comparing results with or without 0.2% sodium hexadecyl sulfate (SHS) added to the gel buffer solution. Images were cropped to show only the regions of interest. Top view: high level overlay showing the relative differences in peak separation efficiency (PSE). Lower views: baseline zoomed-in views highlighting the SHS impact on IP1 resolution from the main RTP-1 peak


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Figure 2. Impact of SHS on the RTP-1 purity method range (linearity). A) Apparent relative proportion of IP1 present with or without SHS in the sieving gel buffer solution. B) IP1 area signal linearity comparison with or without SHS. Note the consistency across the tested concentration range with SHS present. Combined, the data show that without SHS the assay range was limited to peak area signals ≤ 10k relative to ≥ 30k when SHS was used.


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Figure 3. Electropherograms showing the impact of various detergents on RTP-1 PSE (narrowed into the relevant region with a slight offset). Inset: Structures of the detergents added to the gel buffer solutions. The injected RTP-1 sample and gel buffer contained only the detergent noted above the electropherogram. The impact of gel buffer detergent composition on main peak plate count and resolution between the main peak and IP1 are listed in Table 1.


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1.0% SHS 0.2% SHS 4% SDS 2% SDS 1% SDS 0.2% SDS 0.0

5.0

10.0

15.0

Main Peak Plate Count (x10^3)

Figure 4. Effect of varying gel buffer SDS or SHS concentration on RTP-1 main peak plate count. The effect of SDS on plate count reached near saturation at concentrations > 2.0%


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Figure 5. Charge surface profile of the Adnectin portion of RTP-1. Red and blue indicate negative and positive electrostatic charge, respectively. A and B show opposite surface exposed views. Note the high degree of negative charge on the surface shown in A, which may be a barrier to the binding of detergents except for those with longer hydrophobic tails like SHS which can better establish an energetically-favorable interaction with the hydrophobic core of the protein to initiate unfolding.


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Figure 6. SDS:RTP-1 and SHS:RTP-1 Differential Scanning Calorimetry (DSC) profile comparisons. A) Impact of varying the SDS concentration on the RTP-1 DSC profile, and B) the impact of varying the SHS concentration. Two endothermal profiles are clearly visible, E1 and E2, representing the Fc and Adnectin domains respectively, which were assessed for denaturation temperatures (Tm1 and Tm2) and enthalpy (∆H1 and ∆H2). Values for these functions are listed in Table 2. Plots of the enthalpy changes of the two domains against detergent concentration are shown in C and D.


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Figure 7. CGE profiles of other proteins obtained using SDS and SHS gel buffer solutions: mAbs RTP-2 (A) and RTP-3 (B). The light chain and heavy chain peaks are labeled LC and HC respectively. Black and red traces are electropherograms obtained from SDS and SHS gel buffer solutions respectively.


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Figure 8. Comparison of RTP-1 electropherograms obtained with SDS (A), SHS (B) or both (C) in the gel buffer. (C) Inset: Close-up comparison of the 10 kDa I.S. under all three conditions. Note that the addition of SDS to the SHS gel buffer did not significantly impact the PSE profile of RTP-1 (compare panels B and C).


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For Table of Contents Only


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Purity Determination by Capillary Electrophoresis Sodium Hexadecyl

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