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Surface Activity of a Monoclonal Antibody
Surface Activity of a Monoclonal Antibody HANNS-CHRISTIAN MAHLER,1 FRANK SENNER,2 KARSTEN MAEDER,3 ROBERT MUELLER1 1
F. Hoffmann-La Roche Ltd, Formulation R&D Biologics, Pharmaceutical and Analytical R&D, Basel, Switzerland
2
F. Hoffmann-La Roche Ltd, Molecular Properties, Discovery Chemistry, Basel, Switzerland
3
Institute of Pharmaceutical Technology & Biopharmacy, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
Received 3 November 2008; accepted 27 February 2009 Published online 4 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21776
ABSTRACT: The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4525–4533, 2009
Keywords: zation
protein formulation; proteins; protein aggregation; surfactants; stabili-
INTRODUCTION Monoclonal antibodies are prone to a number of physical and chemical degradation routes1,2 and are known for their interaction with interfaces including air–water interfaces which could lead to partial unfolding, successive aggregation, and precipitation.3,4 Nonionic surfactants like polysorbate 20 or 80 are widely used as stabilizers for protein pharmaceuticals against interface-related
Correspondence to: Hanns-Christian Mahler (Telephone: 41-61-68-83174; Fax: 41-61-68-88689; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 4525–4533 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
stress like encountered during shaking or freezethawing.3,5,6 In aqueous solutions surfactants occur in the form of monomers and micelles and have a strong tendency to accumulate at interfaces such as the air–liquid interface. Micelles are fairly monodisperse compact aggregates where the nonpolar groups of the detergent molecules are sequestered into the center and the polar groups face outwards. At low detergent concentrations only monomers occur, whereas at high concentrations free monomers, surface adsorption and micelles exist in equilibrium. The critical micelle concentration (CMC) is a convenient parameter in the description of micelle formation, usually definable within a narrow concentration range. For practical purposes the CMC represents the
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highest monomer detergent concentration (and thereby the highest detergent chemical potential) obtainable.7 A large number of experimental methods exist for determining the value of the CMC. The extensive list of Mukerjee and Mysels8 for instance discusses 71 possibilities for measuring the CMC value. Popular methods are surface tension method (du Nouy ring detachment method, Wilhelmy plate equilibrium), electric conductivity method, light scattering method or spectral change of an additive in the region below and above the CMC. Please refer to Mukerjee and Mysels8 for a detailed discussion of experimental methods for CMC determination. The CMC value does not only depend on environmental conditions like pH, ionic strength and temperature, but also on the experimental set-up and the used method. Nonionic surfactants have been exploited as excipients for their ability to prevent protein denaturation and aggregation. One potential reason for the effectiveness of surfactants is, that they are thermodynamically favored over proteins for adsorption at the interface and displace the protein from the air–water interface.4,9 Polyoxyethylene sorbitan monolaurate (Tween20) and polyoxyethylene sorbitan monooleate (Tween-80), are commonly used nonionic surfactants, which have been demonstrated to protect proteins against aggregation under a variety of conditions, such freezing and thawing,10,11 shaking,3,6 lyophilisation,12 and spray-drying.13 In addition, Tween-20 is used in several marketed parenteral drug products.11,14–16 However, the use of surfactants as excipients may also increase the risk of other protein degradation reactions. For example, surfactant enforced protein aggregation after quiescent longterm storage at elevated temperature has been reported by Treuheit et al.17 Furthermore, protein oxidation induced by surfactant contaminants such as peroxides may be a potential side-effect.18 For these reasons the pharmaceutical scientist should carefully select the optimal surfactant type, concentration, supplier, and storage condition. The need for high concentration antibody formulations increases with indications suggesting self-administration, such as asthma or rheumatoid arthritis.19 The development of high concentration antibody formulations is, however, a major challenge for the formulation scientists.20 Physical characteristics and stability behavior may change significantly with increasing protein
concentration.21–24 The thermodynamic activity of formulation compounds increases exponentially with increasing protein concentration, mainly due to the volume exclusion effect.25 Therefore, the influence of excipients on a high concentration protein pharmaceutical can differ significantly from a low concentration formulation. The protective behavior of surfactants is well-characterized at low protein concentration,6,10,11,26–29 however for high concentration protein formulation the interaction between protein, surfactant and interfaces is still not fully clear. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibodypolysorbate 20 formulation. In a first experiment, the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL was investigated. Shaking is a typical stress, where the protein is exposed to a high level of air–water interfaces and the resulting instabilities can be prevented by addition of a surfactant.3,5,6 For this reason the stability of 10, 75, and 150 mg/mL antibody formulations containing different concentrations of polysorbate 20, either below or above the beforehand determined apparent CMC, was tested in a subsequent shaking stress experiment with regard to their aggregation behavior.
MATERIALS AND METHODS Formulations The monoclonal antibody (Mab) of the isotype IgG1 was formulated in 20 mM L-Histidine/ L-Histidine-HCl (SA Ajinomoto Omnichem NV, Louvain-la-Neuve, Belgium) pH 5.5. The Mab solution was concentrated with a stirred ultrafiltration cell (Millipore, Bedford, MA), equipped with a 30 kDa ultrafiltration membrane (Millipore), to a concentration of approximately 190 mg/mL. All test solutions either containing the pure antibody, the pure polysorbate 20 (Uniquema, Wilton, UK) or mixtures of both, were also formulated in a 20 mM L-histidine-HCl pH 5.5 buffer. For the surface pressure measurements dilution series were prepared by a liquid handling system (Tecan Group Ltd., Ma¨nnedorf, Switzerland, Genesis RSP 150) ranging from 1 mM to 1 mM surface active compound concentration.
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For the shaking stress experiments 0, 10, 75, and 150 mg/mL antibody were formulated into 20 mM L-histidine-HCl pH 5.5 buffer containing 0, 0.005%, 0.01%, 0.02%, or 0.05% (w/v) polysorbate 20. The test formulations were filtered by a 0.2 mm PVDF syringe filter (Millipore) and 1 mL was filled aseptically into 2 mL type I clear glass injection vials (Schott forma vitrum AG, St. Gallen, Switzerland). The vials were closed with a Teflon-coated injection stopper (Daikyo Seiko, Tokyo, Japan) and sealed with an aluminium crimp cap.
Surface Tension and CMC Measurement Surface pressure curves were acquired from a dilution series of pure polysorbate 20 or pure antibody, ranging from 1 mM to 1 mM. For calculation of the dilution series a molecular weight of 150,000 g/mol was used for the antibody and 1228 g/mol for polysorbate 20. In addition, surface pressure curves of polysorbate 20 were measured in the presence of antibody at 10, 37.5, 75, 100, and 150 mg/mL. The CMC was determined from surface pressure versus surfactant concentration curves by application of the Gibbs adsorption isotherm using the software Kibron MultiPi Manager version 2.40. Surface pressure curves were measured by a modified Du Nouy method on a Kibron MultiPi WS 1 multichannel tensiometer. The system was calibrated against water with a known surface tension of 72.5 mNm. All measurements were performed at a temperature of 25 38C. The surface tension of aqueous solutions of surface-active agents decreases rapidly with addition of surfactant until the CMC is reached and then stays constant above the CMC. The Kibron MultiPi WS 1 measures the surface pressure p which is related to the surface tension g by following equation: p ¼ g0 g
(1)
where g0 is the surface tension of the surfactant free solution.
Shaking Stress Study The shaking stress study was performed as described previously.3 Samples were analyzed after 14 days of shaking stress by below described analytical methods. DOI 10.1002/jps
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Analytical Methods Size exclusion chromatography was performed on a Merck Hitachi HPLC system (L-7100 pump, L-7400 UV detector, D-7000 interface, L-7200 autosampler þ peltier sample cooler) equipped with a Tosoh Bioscience TSKgel 3000 SWXL column and a Tosoh Bioscience SWXL guard column. The mobile phase consisted of a 0.2 M dipotassium hydrogen phosphate, 0.25 M potassium chloride pH 7.0 buffer. The separation method ran 30 min with a flow rate of 0.5 mL/min at room temperature. Detection was performed at a wavelength of 280 nm. The samples were stored at 88C in the auto sampler and were injected undiluted onto the column. The injection volume was 20 mL for the 10 mg/mL samples, 5 mL for 75 mg/mL samples and 3 mL for the 150 mg/mL samples. The term high molecular weight (HMW) describes all species, which elute earlier than the main peak, comprising dimers and larger soluble aggregates. Turbidity was determined by light scattering at a wavelength of 350 nm on a Varian Cary 300 UV/ VIS spectrophotometer. The light absorption of the undiluted sample was corrected by subtraction of a water sample. The path length of the quartz cuvette was 1 cm. Visual inspection was carried out using a Seidenader V 90-T (Seidenader Maschinenbau GmbH, Markt Schwaben, Germany). This method allows ‘‘hands-free’’ visible inspection through rotation by rollers. The high concentrated light beams through the bottom, top and side cause particles to reflect light due the Tyndall effect and particles are aided visually through a magnifying lens. The number of particles observed was weighted based on subjective inspection, as (1) free from particles, (2) practically free from particles, (3) several particles, and (4) many particles.
RESULTS Surface pressure curves of pure antibody and polysorbate 20, as well as of antibody-polysorbate mixtures were measured to characterize the influence of high antibody concentration on the surface activity polysorbate 20. Figure 1 compares the surface pressure of pure Mab and polysorbate 20 solutions in 20 mM L-histidine pH 5.5 in a concentration range from 1 mm to 1 mM. Both the antibody and polysorbate show a surface active
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Figure 1. Plots of surface pressure against polysorbate 20 (open squares) concentration and antibody (closed squares) concentration. Both compounds, the surfactant and the monoclonal antibody, are formulated in 20 mM L-histidine-HCl pH 5.5. Surface pressure is measured by a modified Du Nuoy method.
behavior, which can be fitted by a logistic equation of the type f ðxÞ ¼
A1 A2 1 þ ðx=x0 ÞP
þ A2
(2)
where f(x) is the surface pressure and x is the natural logarithm of the surfactant concentration. The parameter A1 is the fitted plateau surface pressure at infinite surfactant dilution. Similarly, A2 represents the fitted plateau surface pressure at the high concentration limit. The parameter x0 measures the surfactant concentration at half maximal surface pressure. Slopes of the sigmoidal curves are related to the exponential P. Although a logistic equation has no special physical or thermodynamic meaning in application to surfactant effects, the fitting procedure provides a statistical means of utilizing data gathered over the entire concentration range in the extraction of fitted parameters with error estimates.30,31 The surface pressure curve of polysorbate 20 shows the full profile of a sigmoidal function, starting with a short flat phase, followed by a linear increase and ended by an asymptotical flattening to 32 mN/m. Whereas the surface pressure curve of the Mab shows a long flat start phase until 10 mM, followed by a linear increase, to a surface pressure of 16 mN/m without reaching a final plateau. This experiment demonstrated that the antibody possesses a significant surface activity at
Figure 2. Normalized surface pressure curves of polysorbate 20 in presence of antibody concentrations increasing from 0 to 150 mg/mL.
concentrations higher than 10 mM, which corresponds to a concentration of 1.5 mg/mL antibody. In the next experiment the influence of the presence of different antibody concentration on the surface activity of polysorbate 20 was tested. Figure 2 shows an overlay of polysorbate 20 surface pressure curves, which were measured in presence of different antibody concentrations, ranging from 0 to 150 mg/mL. The surface pressure values were normalized for clarity by dividing the actual measured values by the maximum surface pressure of the corresponding measurement series for clarification. The overlay of surface pressure curves of the antibody-polysorbate mixtures shows clearly an antibody dependent shift of the end-point of the flat start phase as well as of the start point of the flattening phase to higher surfactant concentration. The flat start phase of the antibody-free polysorbate 20 surface pressure curve ends at 1 mM, whereas in presence of 150 mg/mL antibody the end of lag phase is shifted to 10 mM. From the surface pressure curves of antibodypolysorbate mixtures the critical micelle concentration (CMC) is determined by application of the Gibbs adsorption isotherm. Per definition the Gibbs adsorption isotherm can be applied correctly for a two-component system consisting of a solvent and a surfactant. For that reason the calculated CMC are referred to as apparent CMC values. The CMC is defined as the surfactant concentration, which is necessary to saturate the complete surface. After surface saturation a
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Figure 3. Apparent CMC of polysorbate 20 as a function of antibody concentration (open squares).
further increase of surfactant concentration will lead to the formation of micelles.8 Figure 3 shows the dependence of apparent CMC of polysorbateantibody mixtures as a function of antibody concentration. The curve illustrates clearly, that the presence of antibody shifts the apparent CMC from 0.01% w/v in an antibody-free buffer to almost 0.04% w/v in the presence of 150 mg/mL antibody. The measured CMC value of the antibody-free formulation is in good agreement with previously published CMC values for polysorbate 20 in aqueous solution.7,32,33 Since the CMC of surfactants is discussed to be an important parameter for stabilization of proteins against surface-induced stress like shaking,4,11,34 we tested in a subsequent shaking stress experiment the influence of polysorbate 20 concentration on the stabilization of different concentrations of antibody formulations. Antibody solutions containing 10, 75, or 150 mg/mL Mab were formulated either in a polysorbate-free buffer, or with 0.005%, 0.01%, 0.02%, or 0.05% (w/v) polysorbate 20. The polysorbate concentration were selected to be below or above the measured apparent polysorbate CMC as taken from Figure 3. The samples were filled with 1 mL fill volume into 2 mL vials and were shaken for 14 days at 2–88C. After shaking stress the samples were analyzed by SE-HPLC, turbidity measurement and inspection for visible particles. Figure 4 presents the content of HMW species, as determined by SE-HPLC, of Mab formulations after shaking stress. The results illustrate clearly that the polysorbate concentration did not influence the level of HMW after shaking stress. The DOI 10.1002/jps
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Figure 4. High molecular weight content (HMW) of Mab formulations, as determined by SE-HPLC, depending on the polysorbate 20 level after 2 weeks shaking at 58C.
level of HMW species of unstressed polysorbatefree antibody formulations were 1.7% for 10 mg/ mL, 2.2% for 75 mg/mL, and 2.6% for 150 mg/mL. Comparing HMW of stressed and unstressed polysorbate antibody formulations illustrates that the antibody possesses a very good stability against shaking stress. However, the content of HMW increased significantly with increasing Mab concentration from 1.7% at 10 mg/mL to 2.6% at 150 mg/mL. This observation suggests that increased aggregation is related to the higher antibody concentration and could not be prevented or reduced by addition of polysorbate.
Figure 5. Turbidity of Mab formulations, as determined by A350, as a function of polysorbate 20 level after 2 weeks shaking at 58C.
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Table 1. Visual Appearance of Mab Formulations as a Function of Polysorbate 20 Level After 2 weeks Shaking at 58C Surf Concentration Mab Concentration 0% 0.005% 0.01% 0.02% 0.05% 10 mg/mL 75 mg/mL 150 mg/mL
4a 4b 4c
1 1 1
1 1 1
1 1 1
1 1 1
(1) Free from particles, (2) essentially free from particles, (3) with several particles, (4) with many particles. a Small to medium sized particles. b Fine particles. c Very fine particles.
Figure 5 shows the turbidity of the shaking stressed antibody formulations. As observed in Figure 4 for the HMW results, the turbidity was not influenced by the presence of polysorbate, however it increased with increasing antibody concentration from 0.04 at 10 mg/mL to more than 0.4 at 150 mg/mL. Table 1 summarizes the observations from visual inspection of the shaking stressed samples. In antibody samples which did not contain polysorbate, many visible particles were detected after shaking stress. Depending on the antibody concentration, observed particles differed in size. At 150 mg/mL visible particles appeared to be very fine, whereas at 10 mg/mL visible particles were larger, but less concentrated. The addition of 0.005% (w/v) polysorbate 20 to the antibody formulation was sufficient to avoid the appearance of visible particles in all antibody formulations following shaking stress, independent from the protein concentration.
DISCUSSION Therapeutic proteins are known for their sensitivity against agitation or freeze-thaw stress, which may lead to protein aggregation and precipitation. During shaking the air–water interface is disturbed and the integral structure of protein molecules, which are located in the interfacial region, is continuously damaged.9,35 Partially unfolded protein molecules cluster preferably with each other via hydrophobic interactions, which then lead to aggregation and precipitation of protein molecules.17 A widespread approach to avoid these instabilities is the addition of nonionic surfactants like polysorbate
20 or 80. Surfactants are surface-active molecules which preferably accumulate at the liquid–air or liquid–ice interface and thereby eliminate the protein from the interface. As the stabilization of proteins by surfactants at low protein concentration is well characterized, little is known how surfactants behave at high protein concentration exceeding 100 mg/mL. Therefore, the aim of this study was to characterize the surface behavior of polysorbate over a wide protein concentration range with a model monoclonal IgG1 antibody. Surface activity of proteins is well described in the literature and depends on a variety of solution conditions like protein concentration, pH or ionic strength.9,36 At concentration higher than 10 mM (1.5 mg/mL) the pure antibody increased significantly the surface pressure of the solution, clearly demonstrating surface activity. Comparison of the surface pressure curves of the antibody with the pure polysorbate 20 shows a significant lower surface activity for the antibody. The increase of surface pressure with concentration was smaller for the antibody and the onset was also shifted to higher concentration. In a next experiment the influence of different antibody concentration on the surface activity of polysorbate was tested. The results presented in Figure 2 indicate clearly that the surface activity of the surfactant is influenced by the presence of high antibody concentration. With increasing antibody concentration the normalized surface pressure curve is shifted to higher concentration. Normalization facilitated the comparison of polysorbate surface pressure curves, because the presence of antibody not only shifted the curve, but also decreased overall surface pressure. The normalized curves indicate that the overall form of the surface pressure curve does not change significantly and each curve can be fitted with a logistical sigmoidal equation, which is commonly applicable to surface active behavior.30,31 The overall shift of surface pressure curves to higher concentration suggests that the surface saturation with surfactant molecules is shifted to higher surfactant concentration, which is also illustrated in Figure 3 by the antibody concentration dependent increase of apparent polysorbate 20 CMC from 0.01% in the proteinfree buffer to 0.04% in presence of 150 mg/mL. The application of Gibbs adsorption isotherm for calculating of CMC from surface pressure curves is per definition only allowed for two-component systems consisting of one surface active compound and a solvent. Therefore, calculated CMC values
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from antibody-polysorbate mixture curves are regarded as apparent CMC values. The shift of polysorbate surface tension curves to higher concentrations can be explained by a displacement competition at the air–liquid interface between the surface active antibody and polysorbate molecules. The high concentration of antibody at the interface requires a significant larger quantity of surfactant molecules in order to displace the antibody from the interface in comparison to a protein-free buffer solution. Alternatively, the surfactant may bind to the antibody and therefore requiring larger quantities of surfactant at higher protein concentration to coat the interfacial surface. The CMC and thereby the surfactant concentration needed for complete surface saturation is discussed to correlate with the ability of the formulation to protect the antibody against interface-related stress.4,11,34 Formulations with a surfactant concentration below the CMC should then be more prone to aggregation than formulations with a surfactant concentration above the CMC. Based on this hypothesis a shaking stress experiment was designed, which tested polysorbate concentrations below and above the measured CMC curve at antibody concentrations of 10, 75, and 150 mg/mL. The shaking stress experiment was carried out at a temperature of 2–88C, in order to reduce the influence of potential accompanying chemical degradations. The stressed samples were analyzed by SE-HPLC (soluble aggregates, HMW species), turbidity and inspection on visible particles. The antibody demonstrated good stability against shaking stress at 58C as neither soluble aggregates nor turbidity results show differences between unstressed and stressed samples. An influence of surfactant concentration on antibody stabilization could not be observed by these assays. However, the model antibody clearly underwent physical damage, as the data from visual inspection for protein precipitation showed. This is not an uncommon observation, as other monoclonal antibodies also showed no correlation between the generation of soluble and insoluble aggregates.3 In all surfactant-free antibody formulations, visible particles were detected, but the presence of already 0.005% polysorbate 20, which is even below the CMC of polysorbate 20 in a protein-free solution, was sufficient to prevent the generation of visible particles. Although the apparent CMC of polysorbate 20 is shifted to higher surfactant concentration in the presence of high antibody DOI 10.1002/jps
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concentration, no influence of surfactant concentration on the stabilization of the antibody against shaking stress was observed. The findings of this study deviate surprisingly from some common assumptions. First, the antibody possesses a significant surface activity, however is extremely stable against shaking stress, which deviates from the observations of Levine et al.,9 who reported a positive correlation of surface activity and susceptibility to shakingstressed induced aggregation. A clear explanation for this observation cannot be given, however we assume that beside of surface activity also inherent structural stability governed by covalent and noncovalent bonds play a further key role for stability against interface-related stress. Secondly, although the presence of the antibody shifts the apparent CMC of polysorbate to higher concentrations, a relationship between apparent CMC and the stabilization against shaking stress cannot be observed. For all antibody concentrations 0.005%w/v polysorbate 20, a concentration well below the measured CMC of the pure polysorbate 20 solution, was sufficient to prevent protein aggregation. The independence of stability against shaking stress from apparent CMC can be possibly explained by the self-stabilizing ability of high concentration proteins.5,37 Furthermore, the increased viscosity of high concentration antibody formulations20,22 reduces the introduced mechanical and interfacial stress. Both factors may compensate for the apparent too low surfactant concentration in the protein formulations. The investigated model antibody demonstrated an exceptional good stability against shaking stress, which is from our experience not regarded as typical for other monoclonal antibodies. The characterization of the surface active behavior of further monoclonal antibodies will therefore be subject to comparable studies. Even though SE-HPLC and turbidity analysis were not able to detect a change of aggregation upon shaking stress, both methods were capable of detecting protein concentration dependent aggregation, which could not be prevented or reduced by the addition of surfactant. Interestingly, all antibody formulations, at low and high concentrations, were prepared from the same concentrated stock solution, indicating that the concentration process by ultrafiltration was not responsible for concentration-dependent aggregation. Similarly, turbidity increased as a function of protein concentration. In agreement with
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Sukumar et al.38 the opalescent appearance is a simple consequence of Rayleigh scatter and was not a result of physical instability. Summing-up, the investigated antibody showed interesting formulation properties. Although the model antibody demonstrated a significant surface activity, a high resistance against shaking stress induced aggregation was observed. Furthermore, the antibody influenced the surface activity of polysorbate 20 and shifted the apparent CMC to higher surfactant concentrations. However, the shift of the apparent CMC did not result in a demand for higher surfactant concentrations in order to prevent interface-induced protein aggregation. Surface activity may be an interesting parameter to characterize protein formulations, especially for high protein concentration; however it is not suitable to explain exclusively interface-related aggregation and the role of surfactants in stabilization against this kind of stress.
ACKNOWLEDGMENTS The authors would like to thank Oliver Stauch for his valuable contribution and discussions.
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F. Hoffmann-La Roche Ltd, Formulation R&D Biologics, Pharmaceutical and Analytical R&D, Basel, Switzerland
2
F. Hoffmann-La Roche Ltd, Molecular Properties, Discovery Chemistry, Basel, Switzerland
3
Institute of Pharmaceutical Technology & Biopharmacy, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
Received 3 November 2008; accepted 27 February 2009 Published online 4 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21776
ABSTRACT: The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4525–4533, 2009
Keywords: zation
protein formulation; proteins; protein aggregation; surfactants; stabili-
INTRODUCTION Monoclonal antibodies are prone to a number of physical and chemical degradation routes1,2 and are known for their interaction with interfaces including air–water interfaces which could lead to partial unfolding, successive aggregation, and precipitation.3,4 Nonionic surfactants like polysorbate 20 or 80 are widely used as stabilizers for protein pharmaceuticals against interface-related
Correspondence to: Hanns-Christian Mahler (Telephone: 41-61-68-83174; Fax: 41-61-68-88689; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 4525–4533 (2009) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
stress like encountered during shaking or freezethawing.3,5,6 In aqueous solutions surfactants occur in the form of monomers and micelles and have a strong tendency to accumulate at interfaces such as the air–liquid interface. Micelles are fairly monodisperse compact aggregates where the nonpolar groups of the detergent molecules are sequestered into the center and the polar groups face outwards. At low detergent concentrations only monomers occur, whereas at high concentrations free monomers, surface adsorption and micelles exist in equilibrium. The critical micelle concentration (CMC) is a convenient parameter in the description of micelle formation, usually definable within a narrow concentration range. For practical purposes the CMC represents the
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highest monomer detergent concentration (and thereby the highest detergent chemical potential) obtainable.7 A large number of experimental methods exist for determining the value of the CMC. The extensive list of Mukerjee and Mysels8 for instance discusses 71 possibilities for measuring the CMC value. Popular methods are surface tension method (du Nouy ring detachment method, Wilhelmy plate equilibrium), electric conductivity method, light scattering method or spectral change of an additive in the region below and above the CMC. Please refer to Mukerjee and Mysels8 for a detailed discussion of experimental methods for CMC determination. The CMC value does not only depend on environmental conditions like pH, ionic strength and temperature, but also on the experimental set-up and the used method. Nonionic surfactants have been exploited as excipients for their ability to prevent protein denaturation and aggregation. One potential reason for the effectiveness of surfactants is, that they are thermodynamically favored over proteins for adsorption at the interface and displace the protein from the air–water interface.4,9 Polyoxyethylene sorbitan monolaurate (Tween20) and polyoxyethylene sorbitan monooleate (Tween-80), are commonly used nonionic surfactants, which have been demonstrated to protect proteins against aggregation under a variety of conditions, such freezing and thawing,10,11 shaking,3,6 lyophilisation,12 and spray-drying.13 In addition, Tween-20 is used in several marketed parenteral drug products.11,14–16 However, the use of surfactants as excipients may also increase the risk of other protein degradation reactions. For example, surfactant enforced protein aggregation after quiescent longterm storage at elevated temperature has been reported by Treuheit et al.17 Furthermore, protein oxidation induced by surfactant contaminants such as peroxides may be a potential side-effect.18 For these reasons the pharmaceutical scientist should carefully select the optimal surfactant type, concentration, supplier, and storage condition. The need for high concentration antibody formulations increases with indications suggesting self-administration, such as asthma or rheumatoid arthritis.19 The development of high concentration antibody formulations is, however, a major challenge for the formulation scientists.20 Physical characteristics and stability behavior may change significantly with increasing protein
concentration.21–24 The thermodynamic activity of formulation compounds increases exponentially with increasing protein concentration, mainly due to the volume exclusion effect.25 Therefore, the influence of excipients on a high concentration protein pharmaceutical can differ significantly from a low concentration formulation. The protective behavior of surfactants is well-characterized at low protein concentration,6,10,11,26–29 however for high concentration protein formulation the interaction between protein, surfactant and interfaces is still not fully clear. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibodypolysorbate 20 formulation. In a first experiment, the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL was investigated. Shaking is a typical stress, where the protein is exposed to a high level of air–water interfaces and the resulting instabilities can be prevented by addition of a surfactant.3,5,6 For this reason the stability of 10, 75, and 150 mg/mL antibody formulations containing different concentrations of polysorbate 20, either below or above the beforehand determined apparent CMC, was tested in a subsequent shaking stress experiment with regard to their aggregation behavior.
MATERIALS AND METHODS Formulations The monoclonal antibody (Mab) of the isotype IgG1 was formulated in 20 mM L-Histidine/ L-Histidine-HCl (SA Ajinomoto Omnichem NV, Louvain-la-Neuve, Belgium) pH 5.5. The Mab solution was concentrated with a stirred ultrafiltration cell (Millipore, Bedford, MA), equipped with a 30 kDa ultrafiltration membrane (Millipore), to a concentration of approximately 190 mg/mL. All test solutions either containing the pure antibody, the pure polysorbate 20 (Uniquema, Wilton, UK) or mixtures of both, were also formulated in a 20 mM L-histidine-HCl pH 5.5 buffer. For the surface pressure measurements dilution series were prepared by a liquid handling system (Tecan Group Ltd., Ma¨nnedorf, Switzerland, Genesis RSP 150) ranging from 1 mM to 1 mM surface active compound concentration.
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For the shaking stress experiments 0, 10, 75, and 150 mg/mL antibody were formulated into 20 mM L-histidine-HCl pH 5.5 buffer containing 0, 0.005%, 0.01%, 0.02%, or 0.05% (w/v) polysorbate 20. The test formulations were filtered by a 0.2 mm PVDF syringe filter (Millipore) and 1 mL was filled aseptically into 2 mL type I clear glass injection vials (Schott forma vitrum AG, St. Gallen, Switzerland). The vials were closed with a Teflon-coated injection stopper (Daikyo Seiko, Tokyo, Japan) and sealed with an aluminium crimp cap.
Surface Tension and CMC Measurement Surface pressure curves were acquired from a dilution series of pure polysorbate 20 or pure antibody, ranging from 1 mM to 1 mM. For calculation of the dilution series a molecular weight of 150,000 g/mol was used for the antibody and 1228 g/mol for polysorbate 20. In addition, surface pressure curves of polysorbate 20 were measured in the presence of antibody at 10, 37.5, 75, 100, and 150 mg/mL. The CMC was determined from surface pressure versus surfactant concentration curves by application of the Gibbs adsorption isotherm using the software Kibron MultiPi Manager version 2.40. Surface pressure curves were measured by a modified Du Nouy method on a Kibron MultiPi WS 1 multichannel tensiometer. The system was calibrated against water with a known surface tension of 72.5 mNm. All measurements were performed at a temperature of 25 38C. The surface tension of aqueous solutions of surface-active agents decreases rapidly with addition of surfactant until the CMC is reached and then stays constant above the CMC. The Kibron MultiPi WS 1 measures the surface pressure p which is related to the surface tension g by following equation: p ¼ g0 g
(1)
where g0 is the surface tension of the surfactant free solution.
Shaking Stress Study The shaking stress study was performed as described previously.3 Samples were analyzed after 14 days of shaking stress by below described analytical methods. DOI 10.1002/jps
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Analytical Methods Size exclusion chromatography was performed on a Merck Hitachi HPLC system (L-7100 pump, L-7400 UV detector, D-7000 interface, L-7200 autosampler þ peltier sample cooler) equipped with a Tosoh Bioscience TSKgel 3000 SWXL column and a Tosoh Bioscience SWXL guard column. The mobile phase consisted of a 0.2 M dipotassium hydrogen phosphate, 0.25 M potassium chloride pH 7.0 buffer. The separation method ran 30 min with a flow rate of 0.5 mL/min at room temperature. Detection was performed at a wavelength of 280 nm. The samples were stored at 88C in the auto sampler and were injected undiluted onto the column. The injection volume was 20 mL for the 10 mg/mL samples, 5 mL for 75 mg/mL samples and 3 mL for the 150 mg/mL samples. The term high molecular weight (HMW) describes all species, which elute earlier than the main peak, comprising dimers and larger soluble aggregates. Turbidity was determined by light scattering at a wavelength of 350 nm on a Varian Cary 300 UV/ VIS spectrophotometer. The light absorption of the undiluted sample was corrected by subtraction of a water sample. The path length of the quartz cuvette was 1 cm. Visual inspection was carried out using a Seidenader V 90-T (Seidenader Maschinenbau GmbH, Markt Schwaben, Germany). This method allows ‘‘hands-free’’ visible inspection through rotation by rollers. The high concentrated light beams through the bottom, top and side cause particles to reflect light due the Tyndall effect and particles are aided visually through a magnifying lens. The number of particles observed was weighted based on subjective inspection, as (1) free from particles, (2) practically free from particles, (3) several particles, and (4) many particles.
RESULTS Surface pressure curves of pure antibody and polysorbate 20, as well as of antibody-polysorbate mixtures were measured to characterize the influence of high antibody concentration on the surface activity polysorbate 20. Figure 1 compares the surface pressure of pure Mab and polysorbate 20 solutions in 20 mM L-histidine pH 5.5 in a concentration range from 1 mm to 1 mM. Both the antibody and polysorbate show a surface active
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Figure 1. Plots of surface pressure against polysorbate 20 (open squares) concentration and antibody (closed squares) concentration. Both compounds, the surfactant and the monoclonal antibody, are formulated in 20 mM L-histidine-HCl pH 5.5. Surface pressure is measured by a modified Du Nuoy method.
behavior, which can be fitted by a logistic equation of the type f ðxÞ ¼
A1 A2 1 þ ðx=x0 ÞP
þ A2
(2)
where f(x) is the surface pressure and x is the natural logarithm of the surfactant concentration. The parameter A1 is the fitted plateau surface pressure at infinite surfactant dilution. Similarly, A2 represents the fitted plateau surface pressure at the high concentration limit. The parameter x0 measures the surfactant concentration at half maximal surface pressure. Slopes of the sigmoidal curves are related to the exponential P. Although a logistic equation has no special physical or thermodynamic meaning in application to surfactant effects, the fitting procedure provides a statistical means of utilizing data gathered over the entire concentration range in the extraction of fitted parameters with error estimates.30,31 The surface pressure curve of polysorbate 20 shows the full profile of a sigmoidal function, starting with a short flat phase, followed by a linear increase and ended by an asymptotical flattening to 32 mN/m. Whereas the surface pressure curve of the Mab shows a long flat start phase until 10 mM, followed by a linear increase, to a surface pressure of 16 mN/m without reaching a final plateau. This experiment demonstrated that the antibody possesses a significant surface activity at
Figure 2. Normalized surface pressure curves of polysorbate 20 in presence of antibody concentrations increasing from 0 to 150 mg/mL.
concentrations higher than 10 mM, which corresponds to a concentration of 1.5 mg/mL antibody. In the next experiment the influence of the presence of different antibody concentration on the surface activity of polysorbate 20 was tested. Figure 2 shows an overlay of polysorbate 20 surface pressure curves, which were measured in presence of different antibody concentrations, ranging from 0 to 150 mg/mL. The surface pressure values were normalized for clarity by dividing the actual measured values by the maximum surface pressure of the corresponding measurement series for clarification. The overlay of surface pressure curves of the antibody-polysorbate mixtures shows clearly an antibody dependent shift of the end-point of the flat start phase as well as of the start point of the flattening phase to higher surfactant concentration. The flat start phase of the antibody-free polysorbate 20 surface pressure curve ends at 1 mM, whereas in presence of 150 mg/mL antibody the end of lag phase is shifted to 10 mM. From the surface pressure curves of antibodypolysorbate mixtures the critical micelle concentration (CMC) is determined by application of the Gibbs adsorption isotherm. Per definition the Gibbs adsorption isotherm can be applied correctly for a two-component system consisting of a solvent and a surfactant. For that reason the calculated CMC are referred to as apparent CMC values. The CMC is defined as the surfactant concentration, which is necessary to saturate the complete surface. After surface saturation a
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Figure 3. Apparent CMC of polysorbate 20 as a function of antibody concentration (open squares).
further increase of surfactant concentration will lead to the formation of micelles.8 Figure 3 shows the dependence of apparent CMC of polysorbateantibody mixtures as a function of antibody concentration. The curve illustrates clearly, that the presence of antibody shifts the apparent CMC from 0.01% w/v in an antibody-free buffer to almost 0.04% w/v in the presence of 150 mg/mL antibody. The measured CMC value of the antibody-free formulation is in good agreement with previously published CMC values for polysorbate 20 in aqueous solution.7,32,33 Since the CMC of surfactants is discussed to be an important parameter for stabilization of proteins against surface-induced stress like shaking,4,11,34 we tested in a subsequent shaking stress experiment the influence of polysorbate 20 concentration on the stabilization of different concentrations of antibody formulations. Antibody solutions containing 10, 75, or 150 mg/mL Mab were formulated either in a polysorbate-free buffer, or with 0.005%, 0.01%, 0.02%, or 0.05% (w/v) polysorbate 20. The polysorbate concentration were selected to be below or above the measured apparent polysorbate CMC as taken from Figure 3. The samples were filled with 1 mL fill volume into 2 mL vials and were shaken for 14 days at 2–88C. After shaking stress the samples were analyzed by SE-HPLC, turbidity measurement and inspection for visible particles. Figure 4 presents the content of HMW species, as determined by SE-HPLC, of Mab formulations after shaking stress. The results illustrate clearly that the polysorbate concentration did not influence the level of HMW after shaking stress. The DOI 10.1002/jps
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Figure 4. High molecular weight content (HMW) of Mab formulations, as determined by SE-HPLC, depending on the polysorbate 20 level after 2 weeks shaking at 58C.
level of HMW species of unstressed polysorbatefree antibody formulations were 1.7% for 10 mg/ mL, 2.2% for 75 mg/mL, and 2.6% for 150 mg/mL. Comparing HMW of stressed and unstressed polysorbate antibody formulations illustrates that the antibody possesses a very good stability against shaking stress. However, the content of HMW increased significantly with increasing Mab concentration from 1.7% at 10 mg/mL to 2.6% at 150 mg/mL. This observation suggests that increased aggregation is related to the higher antibody concentration and could not be prevented or reduced by addition of polysorbate.
Figure 5. Turbidity of Mab formulations, as determined by A350, as a function of polysorbate 20 level after 2 weeks shaking at 58C.
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Table 1. Visual Appearance of Mab Formulations as a Function of Polysorbate 20 Level After 2 weeks Shaking at 58C Surf Concentration Mab Concentration 0% 0.005% 0.01% 0.02% 0.05% 10 mg/mL 75 mg/mL 150 mg/mL
4a 4b 4c
1 1 1
1 1 1
1 1 1
1 1 1
(1) Free from particles, (2) essentially free from particles, (3) with several particles, (4) with many particles. a Small to medium sized particles. b Fine particles. c Very fine particles.
Figure 5 shows the turbidity of the shaking stressed antibody formulations. As observed in Figure 4 for the HMW results, the turbidity was not influenced by the presence of polysorbate, however it increased with increasing antibody concentration from 0.04 at 10 mg/mL to more than 0.4 at 150 mg/mL. Table 1 summarizes the observations from visual inspection of the shaking stressed samples. In antibody samples which did not contain polysorbate, many visible particles were detected after shaking stress. Depending on the antibody concentration, observed particles differed in size. At 150 mg/mL visible particles appeared to be very fine, whereas at 10 mg/mL visible particles were larger, but less concentrated. The addition of 0.005% (w/v) polysorbate 20 to the antibody formulation was sufficient to avoid the appearance of visible particles in all antibody formulations following shaking stress, independent from the protein concentration.
DISCUSSION Therapeutic proteins are known for their sensitivity against agitation or freeze-thaw stress, which may lead to protein aggregation and precipitation. During shaking the air–water interface is disturbed and the integral structure of protein molecules, which are located in the interfacial region, is continuously damaged.9,35 Partially unfolded protein molecules cluster preferably with each other via hydrophobic interactions, which then lead to aggregation and precipitation of protein molecules.17 A widespread approach to avoid these instabilities is the addition of nonionic surfactants like polysorbate
20 or 80. Surfactants are surface-active molecules which preferably accumulate at the liquid–air or liquid–ice interface and thereby eliminate the protein from the interface. As the stabilization of proteins by surfactants at low protein concentration is well characterized, little is known how surfactants behave at high protein concentration exceeding 100 mg/mL. Therefore, the aim of this study was to characterize the surface behavior of polysorbate over a wide protein concentration range with a model monoclonal IgG1 antibody. Surface activity of proteins is well described in the literature and depends on a variety of solution conditions like protein concentration, pH or ionic strength.9,36 At concentration higher than 10 mM (1.5 mg/mL) the pure antibody increased significantly the surface pressure of the solution, clearly demonstrating surface activity. Comparison of the surface pressure curves of the antibody with the pure polysorbate 20 shows a significant lower surface activity for the antibody. The increase of surface pressure with concentration was smaller for the antibody and the onset was also shifted to higher concentration. In a next experiment the influence of different antibody concentration on the surface activity of polysorbate was tested. The results presented in Figure 2 indicate clearly that the surface activity of the surfactant is influenced by the presence of high antibody concentration. With increasing antibody concentration the normalized surface pressure curve is shifted to higher concentration. Normalization facilitated the comparison of polysorbate surface pressure curves, because the presence of antibody not only shifted the curve, but also decreased overall surface pressure. The normalized curves indicate that the overall form of the surface pressure curve does not change significantly and each curve can be fitted with a logistical sigmoidal equation, which is commonly applicable to surface active behavior.30,31 The overall shift of surface pressure curves to higher concentration suggests that the surface saturation with surfactant molecules is shifted to higher surfactant concentration, which is also illustrated in Figure 3 by the antibody concentration dependent increase of apparent polysorbate 20 CMC from 0.01% in the proteinfree buffer to 0.04% in presence of 150 mg/mL. The application of Gibbs adsorption isotherm for calculating of CMC from surface pressure curves is per definition only allowed for two-component systems consisting of one surface active compound and a solvent. Therefore, calculated CMC values
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from antibody-polysorbate mixture curves are regarded as apparent CMC values. The shift of polysorbate surface tension curves to higher concentrations can be explained by a displacement competition at the air–liquid interface between the surface active antibody and polysorbate molecules. The high concentration of antibody at the interface requires a significant larger quantity of surfactant molecules in order to displace the antibody from the interface in comparison to a protein-free buffer solution. Alternatively, the surfactant may bind to the antibody and therefore requiring larger quantities of surfactant at higher protein concentration to coat the interfacial surface. The CMC and thereby the surfactant concentration needed for complete surface saturation is discussed to correlate with the ability of the formulation to protect the antibody against interface-related stress.4,11,34 Formulations with a surfactant concentration below the CMC should then be more prone to aggregation than formulations with a surfactant concentration above the CMC. Based on this hypothesis a shaking stress experiment was designed, which tested polysorbate concentrations below and above the measured CMC curve at antibody concentrations of 10, 75, and 150 mg/mL. The shaking stress experiment was carried out at a temperature of 2–88C, in order to reduce the influence of potential accompanying chemical degradations. The stressed samples were analyzed by SE-HPLC (soluble aggregates, HMW species), turbidity and inspection on visible particles. The antibody demonstrated good stability against shaking stress at 58C as neither soluble aggregates nor turbidity results show differences between unstressed and stressed samples. An influence of surfactant concentration on antibody stabilization could not be observed by these assays. However, the model antibody clearly underwent physical damage, as the data from visual inspection for protein precipitation showed. This is not an uncommon observation, as other monoclonal antibodies also showed no correlation between the generation of soluble and insoluble aggregates.3 In all surfactant-free antibody formulations, visible particles were detected, but the presence of already 0.005% polysorbate 20, which is even below the CMC of polysorbate 20 in a protein-free solution, was sufficient to prevent the generation of visible particles. Although the apparent CMC of polysorbate 20 is shifted to higher surfactant concentration in the presence of high antibody DOI 10.1002/jps
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concentration, no influence of surfactant concentration on the stabilization of the antibody against shaking stress was observed. The findings of this study deviate surprisingly from some common assumptions. First, the antibody possesses a significant surface activity, however is extremely stable against shaking stress, which deviates from the observations of Levine et al.,9 who reported a positive correlation of surface activity and susceptibility to shakingstressed induced aggregation. A clear explanation for this observation cannot be given, however we assume that beside of surface activity also inherent structural stability governed by covalent and noncovalent bonds play a further key role for stability against interface-related stress. Secondly, although the presence of the antibody shifts the apparent CMC of polysorbate to higher concentrations, a relationship between apparent CMC and the stabilization against shaking stress cannot be observed. For all antibody concentrations 0.005%w/v polysorbate 20, a concentration well below the measured CMC of the pure polysorbate 20 solution, was sufficient to prevent protein aggregation. The independence of stability against shaking stress from apparent CMC can be possibly explained by the self-stabilizing ability of high concentration proteins.5,37 Furthermore, the increased viscosity of high concentration antibody formulations20,22 reduces the introduced mechanical and interfacial stress. Both factors may compensate for the apparent too low surfactant concentration in the protein formulations. The investigated model antibody demonstrated an exceptional good stability against shaking stress, which is from our experience not regarded as typical for other monoclonal antibodies. The characterization of the surface active behavior of further monoclonal antibodies will therefore be subject to comparable studies. Even though SE-HPLC and turbidity analysis were not able to detect a change of aggregation upon shaking stress, both methods were capable of detecting protein concentration dependent aggregation, which could not be prevented or reduced by the addition of surfactant. Interestingly, all antibody formulations, at low and high concentrations, were prepared from the same concentrated stock solution, indicating that the concentration process by ultrafiltration was not responsible for concentration-dependent aggregation. Similarly, turbidity increased as a function of protein concentration. In agreement with
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Sukumar et al.38 the opalescent appearance is a simple consequence of Rayleigh scatter and was not a result of physical instability. Summing-up, the investigated antibody showed interesting formulation properties. Although the model antibody demonstrated a significant surface activity, a high resistance against shaking stress induced aggregation was observed. Furthermore, the antibody influenced the surface activity of polysorbate 20 and shifted the apparent CMC to higher surfactant concentrations. However, the shift of the apparent CMC did not result in a demand for higher surfactant concentrations in order to prevent interface-induced protein aggregation. Surface activity may be an interesting parameter to characterize protein formulations, especially for high protein concentration; however it is not suitable to explain exclusively interface-related aggregation and the role of surfactants in stabilization against this kind of stress.
ACKNOWLEDGMENTS The authors would like to thank Oliver Stauch for his valuable contribution and discussions.
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