Pharmaceutical Development and Technology
ISSN: 1083-7450 (Print) 1097-9867 (Online) Journal homepage: http://www.tandfonline.com/loi/iphd20
Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80 Jakson Clark, Jade Montgomery, Ryan Squires & Joseph McGuire To cite this article: Jakson Clark, Jade Montgomery, Ryan Squires & Joseph McGuire (2014): Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80, Pharmaceutical Development and Technology To link to this article: http://dx.doi.org/10.3109/10837450.2014.987297
Published online: 04 Dec 2014.
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Date: 08 November 2015, At: 13:41
http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.987297
TECHNICAL NOTE
Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80 Jakson Clark, Jade Montgomery, Ryan Squires, and Joseph McGuire
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, USA
Abstract
Keywords
Detection of enhanced surface tension depression by surfactant in the presence of protein was recently suggested as a basis for determining whether protein stabilization by that surfactant is owing to surfactant forming a steric barrier at interfaces or surfactant association with the protein. In particular, protein interaction with surfactant aggregates may lead to an increased concentration of monomers thus enhancing surfactant adsorption, or to formation of surfactant–protein complexes having little or no effect on adsorption. We compared the initial rates of surface tension depression by poloxamer 188 and polysorbate 80 (PS 80) in the presence and absence of a human recombinant factor VIII (rFVIII). Indirect evidence had suggested poloxamer 188 enters into stable associations with rFVIII in solution but does not form a steric barrier at the interface, while PS 80 behaves in contrary fashion. In this study, we show the presence of rFVIII caused an increase in the rate (reduction in the activation energy) of PS 80 adsorption, while no such change was recorded in the case of poloxamer 188. Thus, we provide substantiation for detection of protein-mediated acceleration of surfactant adsorption as a means to compare different surfactants in relation to their favored mechanism for protein stabilization.
Accelerated surfactant adsorption, rate analysis, surface tension depression, surfactant selection
Introduction Surfactants stabilize therapeutic proteins by their preferential location at interfaces, inhibiting protein adsorption, and/or association with protein molecules, inhibiting adsorption as well as aggregation1–3. In general, surfactant–protein association must be at play for effective stabilization against activity loss. Current selection of surfactants for therapeutic protein stabilization is not made with any deliberate assurance that this condition is met. Joshi et al2. studied the adsorption, structural alteration and biological activity of a recombinant factor VIII (rFVIII) in the presence of polysorbate 80 (PS 80) at solid surfaces. They found PS 80 effectively prevented rFVIII adsorption at hydrophobic surfaces, but only by PS 80 blocking the interface and not by its association with rFVIII molecules. These conclusions were corroborated through later study of the rFVIII-PS 80 system using air–water surface tension kinetic measurements4. Poloxamer 188 is mainly used upstream for its stabilization of mammalian cells against hydrodynamic stress in cell culture. In a study of its effect on protein adsorption, Kim et al.5 reported adsorption of rFVIII to air–water interfaces was substantially reduced in the presence of poloxamer 188, even at poloxamer concentrations several orders of magnitude below the Critical Micelle Concentration (CMC). Similarly, in tests with solid
Address for correspondence: Joseph McGuire, School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, USA. Tel: 541-737-6306. Fax: 541-737-4600. E-mail: [email protected]
History Received 18 August 2014 Revised 20 October 2014 Accepted 29 October 2014 Published online 3 December 2014
surfaces for which rFVIII affinity is high while poloxamer 188 affinity is very low, they recorded substantially reduced surface adsorption of rFVIII in the presence of poloxamer 188. Taken together, these results2,4,5 indicate that PS 80 did not participate in stable associations with rFVIII in solution. In contrast, poloxamer 188, with comparatively little affinity for hydrophobic association at air–water or synthetic solid–water interfaces, was able to enter into stable associations with rFVIII in solution and in this way stabilize the protein against adsorption loss. The kinetics of surface tension depression of surfactant– protein mixtures is sometimes greater than that recorded for surfactant alone at the same concentration, and this has been observed in all of our previous work with polysorbate-protein mixtures4,6,7. We expected that such ‘‘accelerated adsorption’’ is governed by surfactant–protein interactions and must therefore be a function of parameters important to protein stabilization by that surfactant. In particular, the adsorption of polysorbate monomers may be enhanced by interaction between surfactant aggregates and protein near the interface, leading to the release of monomers for adsorption. We hypothesized that parameters determining whether surfactant stabilization occurs mainly by association with the interface or by association with protein may be quantifiable for different surfactants by analysis of such accelerated adsorption data6,7. As depicted in Figure 1 (top), transport of monomers from an aggregated state in the sublayer to the air–water interface in the absence of protein requires dissociation from the aggregate and monomer migration through the liquid to adsorb. The presence of protein may facilitate aggregate disruption, leading either to an increased concentration of surfactant monomers that would enhance the adsorption rate (Figure 1, middle), or to the formation of surfactant–protein complexes having little or no
J. Clark et al.
Pharm Dev Technol, Early Online: 1–5
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
2
Figure 1. Proposed mechanism of surfactant adsorption to the air–water interface in the absence of protein (top) and in the presence of protein where surfactant preferentially locates at the interface (middle) or where surfactant preferentially associates with the protein (bottom).
effect on surfactant adsorption rate (Figure 1, bottom). It is reasonable to expect that accelerated surfactant adsorption in the presence of protein will occur with surfactants that stabilize protein mainly by preferential location at interfaces, while accelerated surfactant adsorption will not occur with surfactants that form stable surfactant–protein associations. In this article, the kinetics of surface tension depression exhibited by PS 80 and poloxamer 188 in the presence and absence of rFVIII are interpreted with reference to the mechanism of Figure 1. This interpretation provides a rationale for linking accelerated surfactant adsorption outcomes to the dominant mode of rFVIII stabilization by that surfactant (i.e. whether by association with the interface or by association with the protein).
Methods Sample preparation The rFVIII was a gift from Bayer HealthCare (Berkeley, CA), supplied in vials containing 2000 IU of freeze-dried, finished product (5 IU is equivalent to about 1 mg of active protein). The powder was first dissolved in 5.0 mL HPLC water, resulting in a solution containing rFVIII at about 400 IU/mL in ‘‘KG-2’’ buffer (consisting of 30 mM NaCl, 2.5 mM CaCl2, 22 g/L glycine, 3.1 g/L L-histidine, 10 g/L sucrose and 80 mg/L PS 80). The rFVIII solution was then passed through a 10 kDa exclusion filter (Centriplus, Amicon, Darmstadt, Germany) at 3000 Relative Centrifugal Force (RCF) for 1.5 h at 4 C to bring the sample to
DOI: 10.3109/10837450.2014.987297
Stabilization of a human recombinant factor VIII by poloxamer 188
1 mL. rFVIII solutions were used immediately after preparation. PS 80 was prepared in stock injection concentrations of 1000, 2000 and 3000 mg/L, while poloxamer 188 was prepared in stock injection concentrations of 200, 1000 and 2000 mg/L.
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Interfacial tensiometry The surface tension kinetics of PS 80 at 50, 100 and 150 mg/L and poloxamer 188 at 10, 50 and 100 mg/L at 4, 20 and 40 C with and without rFVIII were recorded over a period of about 300 s using a ˚ T10, computer-controlled, automatic tensiometer (Model FTA First Ten Angstroms, Portsmouth, VA). We used the corrected Du Nou¨y ring method of interfacial surface tension measurement in the work described in this study. Sample temperature was controlled by mechanisms constructed in-house. Desired surfactant stock (1.0 mL)was injected into 19 mL (polysorbate-free) KG-2 buffer for trials with surfactant in the absence of protein. Equal 1.0 mL volumes of stock surfactant and filtered rFVIII solutions were combined and injected into 18 mL of (polysorbatefree) KG-2 buffer for trials with surfactant in the presence of protein. All experiments were performed in triplicate. The rFVIII concentration in each sample was determined immediately after completion of each surface tension measurement by detection of UV absorbance at 280 nm. rFVIII concentration ranged from 69 to 122 IU/mL (about 14–24 mg total protein/mL) in tests with PS 80, and 82 to 119 IU/mL (about 16–24 mg total protein/mL) in tests with poloxamer 188.
Results Protein effects on the rate of surface tension depression by surfactant In order to determine the initial rate in each case, surface tension versus time (t) data were fit to a simple three-parameter exponential decay function, i.e. b f ðtÞ¼ae tþc as illustrated in Figure 2 (for adsorption of PS 80 (50 mg/L, 40 C) with and without added rFVIII). Initial slopes were then determined by the derivative of the decay function at the time of injection, t ¼ 0, for each triplicate data set.
Figure 2. Enhanced surface tension depression (adsorption) of 50 ppm PS 80 at 40 C observed upon in the presence of rFVIII.
3
The initial rates of surface tension depression for PS 80 and poloxamer 188, with and without rFVIII, are presented in Figure 3. Initial rates recorded in the presence of rFVIII are displayed as white bars at each temperature; thus white regions visible in the upper portion of any bar represent trials in which the rate of surface tension depression was increased with addition of rFVIII. Accelerated surfactant adsorption in the presence of rFVIII was recorded in seven of nine trials for PS 80, while accelerated surfactant adsorption in the presence of rFVIII was recorded in only two of nine trials for poloxamer 188. Experimental uncertainty was generally large, such that the accelerated adsorption recorded for PS 80 in the presence of rFVIII was statistically significant in only three of the seven instances at a 95% confidence level (50 mg/L PS 80 at 40 C and 150 mg/L PS 80 at 4 and 20 C). The two instances of accelerated adsorption recorded for poloxamer 188 in the presence of rFVIII were not statistically significant. Analysis of the rate data The initial rates of surface tension depression recorded for each surfactant were used to determine a rate constant (km) and reaction order (n) at each temperature (T), i.e. initial rate¼km C n where C is surfactant concentration. The activation energy, Ea, for adsorption in each case was derived from the slope of a plot of ln km versus T1 , after Arrhenius expansion of the rate constant, i.e. Ea 1 þ lnð AÞ lnðkm Þ¼ R T where R is the gas constant and A is the Arrhenius preexponential factor (Figure 4). The activation energies determined in this way for adsorption of PS 80 with and without rFVIII were 6.8 ± 6.1 kJ/mol and 8.4 ± 1.7 kJ/mol, respectively. The activation energies for adsorption of poloxamer 188 with and without rFVIII were determined to be 29.4 ± 15.1 kJ/mol and 18.6 ± 1.1 kJ/mol, respectively (errors are based on the sample variation in slopes within the 95% confidence interval in each case). The presence of protein reduced the activation energy for adsorption of PS 80, but did not reduce the activation energy for adsorption of poloxamer 188.
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Pharm Dev Technol, Early Online: 1–5
Figure 3. Initial rate of surface tension depression of PS 80 (left) and poloxamer 188 (right) with and without rFVIII. Trials conducted with rFVIII represented as blank fill, trials without rFVIII represented as colored fill. Error bars represent 95% confidence levels.
Discussion The decrease in Ea for PS 80 adsorption in the presence of rFVIII is consistent with the mechanism depicted in the middle panel of Figure 1, while the absence of such a decrease in Ea for poloxamer 188 adsorption is consistent with the mechanism depicted in the bottom panel of Figure 1. Qualitatively, these results support the notion that PS 80 has a stronger affinity for the air–water interface than for hydrophobic regions on rFVIII, while poloxamer 188 preferentially adsorbs to rFVIII rather than the air-water interface. Additional justification for this notion lies in the fact that the presence of salt is known to lower the critical aggregation concentration of triblock polymers containing PEO or other polyethers8, and the surface at an air–water interface is depleted of salts relative to the bulk solution9. Thus our results provide a rationale for results recorded by Joshi et al. and Kim et al. showing poloxamer 188 to more effectively stabilize rFVIII against aggregation in solution, as well as adsorption loss, relative to PS 80. Our analysis of rate data is based on an equilibrium between the intermediate species and the products in the reaction schemes depicted in the middle and bottom panels of Figure 1, i.e. based on conversion of reactants to the structural intermediate being rate-limiting. Schram & Hall10 carried out this kind of analysis in study of the effect of selected hydrophobic proteins on pulmonary surfactant function, as did Kim et al.7 in study of the effect of lysozyme and a recombinant protein drug on polysorbate and poloxamer adsorption. It is worth noting they also considered the
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Figure 4. Arrhenius plots for poloxamer 188 and PS 80 with and without rFVIII. Activation energy of surfactant adsorption to the interface is calculated using the slope (¼Ea/R). Error bars represent 95% confidence levels.
alternative case, assuming equilibrium between reactants and the structural intermediate, in which case the second step in each scheme (conversion of structural intermediate to products) is the rate-limiting step. In this case, known as ‘‘transition state theory’’11, the rate constant is independent of the nature of the structural intermediate and is defined absolutely as km ¼constant
K b T DG=RT e h
where kb and h are Boltzmann’s and Planck’s constants, respectively, and DG is the free energy of transition. Since DG ¼ DH T DS, the temperature effect on km is well defined and given by ln km DH 1 ln kb DS þconstant þ ¼ þ R T R T h The slope and intercept of plots of ln km/T versus 1/T would therefore provide estimates of the enthalpy (DH) and entropy (DS) of the transition, and DG can thus be calculated at any desired temperature. Invoking transition state theory to describe the temperature effect on protein-mediated acceleration of surfactant adsorption, Schram & Hall as well as Kim et al. found any reduction in DG observed upon addition of protein was owing entirely to a reduction in DH, suggesting that protein is accelerating the adsorption of the surfactants not by reducing the entropic barrier faced by the surfactant in moving from the aggregate to the interface, but by disrupting the surfactant self-association.
DOI: 10.3109/10837450.2014.987297
Stabilization of a human recombinant factor VIII by poloxamer 188
Conclusions Our results are consistent with those of earlier reports suggesting that characterization of protein-mediated acceleration of surfactant adsorption could be used to quantitatively compare different surfactants in relation to their favored mechanism for imparting protein stability and to guide development of surfactant chemistries optimized for protein stabilization under specific conditions. This kind of approach to surfactant selection and design for reduction of aggregation and adsorption loss does not currently exist, and we provide in this study a rationale for hypotheses to drive further discovery and understanding in this important area.
Acknowledgements We thank Dr Omkar Joshi of Bayer Healthcare, LLC, Berkeley, CA, for providing the rFVIII.
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Declaration of interest We report no declaration of interest.
References 1. Bee J, Randolph T, Carpenter J, et al. Effects of surfaces and leachables on the stability of biopharmaceuticals. J Pharm Sci 2011; 100:4158–4170.
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2. Joshi O, McGuire J, Wang D. Adsorption and function of recombinant factor VIII at solid-water interfaces in the presence of Tween-80. J Pharm Sci 2008;97:4741–4755. 3. Chou D, Krishnamurthy R, Randolph T, et al. Effects of Tween 20 and Tween 80 on the stability of albutropin during agitation. J Pharm Sci 2005;94:1368–1381. 4. Joshi O, Chu L, McGuire J, Wang, D. Adsorption and function of recombinant Factor VIII at the air-water interface in the presence of Tween 80. J Pharm Sci 2009;98:3099–3107. 5. Kim H, McAuley A, Livesay B, et al. Modulation of protein adsorption by poloxamer 188 in relation to polysorbates 80 and 20 at solid surfaces. J Pharm Sci 2014;103:1043–1049. 6. Lee H, McAuley A, Schilke K, McGuire J. Molecular origins of surfactant-mediated stabilization of protein drugs. Adv Drug Deliv Rev 2011;63:1160–1171. 7. Kim H, McAuley A, McGuire J. Protein effects on surfactant adsorption suggest the dominant mode of surfactant-mediated stabilization of protein. J Pharm Sci 2014;103:1337–1345. 8. Patel K, Bharatiya B, Kadam Y, Bahadur P. Micellization and clouding behavior of EO–PO block copolymer in aqueous salt solutions. J Surfactants Deterg 2010;13:89–95. 9. Levin Y, dos Santos A, Diehl A. Ions at the air-water interface: an end to a hundred-year-old mystery? Phys Rev Lett 2009;103: 257802–257805. 10. Schram V, Hall S. Thermodynamic effects of the hydrophobic surfactant proteins on the early adsorption of pulmonary surfactant. Biophys J 2001;81:1536–1546. 11. Levenspiel O. Chemical reaction engineering. 3rd ed. New York: Wiley; 1999:24–26.
ISSN: 1083-7450 (Print) 1097-9867 (Online) Journal homepage: http://www.tandfonline.com/loi/iphd20
Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80 Jakson Clark, Jade Montgomery, Ryan Squires & Joseph McGuire To cite this article: Jakson Clark, Jade Montgomery, Ryan Squires & Joseph McGuire (2014): Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80, Pharmaceutical Development and Technology To link to this article: http://dx.doi.org/10.3109/10837450.2014.987297
Published online: 04 Dec 2014.
Submit your article to this journal
Article views: 40
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphd20 Download by: [University of Lethbridge]
Date: 08 November 2015, At: 13:41
http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.987297
TECHNICAL NOTE
Stabilization of a human recombinant factor VIII by poloxamer 188 in relation to polysorbate 80 Jakson Clark, Jade Montgomery, Ryan Squires, and Joseph McGuire
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, USA
Abstract
Keywords
Detection of enhanced surface tension depression by surfactant in the presence of protein was recently suggested as a basis for determining whether protein stabilization by that surfactant is owing to surfactant forming a steric barrier at interfaces or surfactant association with the protein. In particular, protein interaction with surfactant aggregates may lead to an increased concentration of monomers thus enhancing surfactant adsorption, or to formation of surfactant–protein complexes having little or no effect on adsorption. We compared the initial rates of surface tension depression by poloxamer 188 and polysorbate 80 (PS 80) in the presence and absence of a human recombinant factor VIII (rFVIII). Indirect evidence had suggested poloxamer 188 enters into stable associations with rFVIII in solution but does not form a steric barrier at the interface, while PS 80 behaves in contrary fashion. In this study, we show the presence of rFVIII caused an increase in the rate (reduction in the activation energy) of PS 80 adsorption, while no such change was recorded in the case of poloxamer 188. Thus, we provide substantiation for detection of protein-mediated acceleration of surfactant adsorption as a means to compare different surfactants in relation to their favored mechanism for protein stabilization.
Accelerated surfactant adsorption, rate analysis, surface tension depression, surfactant selection
Introduction Surfactants stabilize therapeutic proteins by their preferential location at interfaces, inhibiting protein adsorption, and/or association with protein molecules, inhibiting adsorption as well as aggregation1–3. In general, surfactant–protein association must be at play for effective stabilization against activity loss. Current selection of surfactants for therapeutic protein stabilization is not made with any deliberate assurance that this condition is met. Joshi et al2. studied the adsorption, structural alteration and biological activity of a recombinant factor VIII (rFVIII) in the presence of polysorbate 80 (PS 80) at solid surfaces. They found PS 80 effectively prevented rFVIII adsorption at hydrophobic surfaces, but only by PS 80 blocking the interface and not by its association with rFVIII molecules. These conclusions were corroborated through later study of the rFVIII-PS 80 system using air–water surface tension kinetic measurements4. Poloxamer 188 is mainly used upstream for its stabilization of mammalian cells against hydrodynamic stress in cell culture. In a study of its effect on protein adsorption, Kim et al.5 reported adsorption of rFVIII to air–water interfaces was substantially reduced in the presence of poloxamer 188, even at poloxamer concentrations several orders of magnitude below the Critical Micelle Concentration (CMC). Similarly, in tests with solid
Address for correspondence: Joseph McGuire, School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, USA. Tel: 541-737-6306. Fax: 541-737-4600. E-mail: [email protected]
History Received 18 August 2014 Revised 20 October 2014 Accepted 29 October 2014 Published online 3 December 2014
surfaces for which rFVIII affinity is high while poloxamer 188 affinity is very low, they recorded substantially reduced surface adsorption of rFVIII in the presence of poloxamer 188. Taken together, these results2,4,5 indicate that PS 80 did not participate in stable associations with rFVIII in solution. In contrast, poloxamer 188, with comparatively little affinity for hydrophobic association at air–water or synthetic solid–water interfaces, was able to enter into stable associations with rFVIII in solution and in this way stabilize the protein against adsorption loss. The kinetics of surface tension depression of surfactant– protein mixtures is sometimes greater than that recorded for surfactant alone at the same concentration, and this has been observed in all of our previous work with polysorbate-protein mixtures4,6,7. We expected that such ‘‘accelerated adsorption’’ is governed by surfactant–protein interactions and must therefore be a function of parameters important to protein stabilization by that surfactant. In particular, the adsorption of polysorbate monomers may be enhanced by interaction between surfactant aggregates and protein near the interface, leading to the release of monomers for adsorption. We hypothesized that parameters determining whether surfactant stabilization occurs mainly by association with the interface or by association with protein may be quantifiable for different surfactants by analysis of such accelerated adsorption data6,7. As depicted in Figure 1 (top), transport of monomers from an aggregated state in the sublayer to the air–water interface in the absence of protein requires dissociation from the aggregate and monomer migration through the liquid to adsorb. The presence of protein may facilitate aggregate disruption, leading either to an increased concentration of surfactant monomers that would enhance the adsorption rate (Figure 1, middle), or to the formation of surfactant–protein complexes having little or no
J. Clark et al.
Pharm Dev Technol, Early Online: 1–5
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
2
Figure 1. Proposed mechanism of surfactant adsorption to the air–water interface in the absence of protein (top) and in the presence of protein where surfactant preferentially locates at the interface (middle) or where surfactant preferentially associates with the protein (bottom).
effect on surfactant adsorption rate (Figure 1, bottom). It is reasonable to expect that accelerated surfactant adsorption in the presence of protein will occur with surfactants that stabilize protein mainly by preferential location at interfaces, while accelerated surfactant adsorption will not occur with surfactants that form stable surfactant–protein associations. In this article, the kinetics of surface tension depression exhibited by PS 80 and poloxamer 188 in the presence and absence of rFVIII are interpreted with reference to the mechanism of Figure 1. This interpretation provides a rationale for linking accelerated surfactant adsorption outcomes to the dominant mode of rFVIII stabilization by that surfactant (i.e. whether by association with the interface or by association with the protein).
Methods Sample preparation The rFVIII was a gift from Bayer HealthCare (Berkeley, CA), supplied in vials containing 2000 IU of freeze-dried, finished product (5 IU is equivalent to about 1 mg of active protein). The powder was first dissolved in 5.0 mL HPLC water, resulting in a solution containing rFVIII at about 400 IU/mL in ‘‘KG-2’’ buffer (consisting of 30 mM NaCl, 2.5 mM CaCl2, 22 g/L glycine, 3.1 g/L L-histidine, 10 g/L sucrose and 80 mg/L PS 80). The rFVIII solution was then passed through a 10 kDa exclusion filter (Centriplus, Amicon, Darmstadt, Germany) at 3000 Relative Centrifugal Force (RCF) for 1.5 h at 4 C to bring the sample to
DOI: 10.3109/10837450.2014.987297
Stabilization of a human recombinant factor VIII by poloxamer 188
1 mL. rFVIII solutions were used immediately after preparation. PS 80 was prepared in stock injection concentrations of 1000, 2000 and 3000 mg/L, while poloxamer 188 was prepared in stock injection concentrations of 200, 1000 and 2000 mg/L.
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Interfacial tensiometry The surface tension kinetics of PS 80 at 50, 100 and 150 mg/L and poloxamer 188 at 10, 50 and 100 mg/L at 4, 20 and 40 C with and without rFVIII were recorded over a period of about 300 s using a ˚ T10, computer-controlled, automatic tensiometer (Model FTA First Ten Angstroms, Portsmouth, VA). We used the corrected Du Nou¨y ring method of interfacial surface tension measurement in the work described in this study. Sample temperature was controlled by mechanisms constructed in-house. Desired surfactant stock (1.0 mL)was injected into 19 mL (polysorbate-free) KG-2 buffer for trials with surfactant in the absence of protein. Equal 1.0 mL volumes of stock surfactant and filtered rFVIII solutions were combined and injected into 18 mL of (polysorbatefree) KG-2 buffer for trials with surfactant in the presence of protein. All experiments were performed in triplicate. The rFVIII concentration in each sample was determined immediately after completion of each surface tension measurement by detection of UV absorbance at 280 nm. rFVIII concentration ranged from 69 to 122 IU/mL (about 14–24 mg total protein/mL) in tests with PS 80, and 82 to 119 IU/mL (about 16–24 mg total protein/mL) in tests with poloxamer 188.
Results Protein effects on the rate of surface tension depression by surfactant In order to determine the initial rate in each case, surface tension versus time (t) data were fit to a simple three-parameter exponential decay function, i.e. b f ðtÞ¼ae tþc as illustrated in Figure 2 (for adsorption of PS 80 (50 mg/L, 40 C) with and without added rFVIII). Initial slopes were then determined by the derivative of the decay function at the time of injection, t ¼ 0, for each triplicate data set.
Figure 2. Enhanced surface tension depression (adsorption) of 50 ppm PS 80 at 40 C observed upon in the presence of rFVIII.
3
The initial rates of surface tension depression for PS 80 and poloxamer 188, with and without rFVIII, are presented in Figure 3. Initial rates recorded in the presence of rFVIII are displayed as white bars at each temperature; thus white regions visible in the upper portion of any bar represent trials in which the rate of surface tension depression was increased with addition of rFVIII. Accelerated surfactant adsorption in the presence of rFVIII was recorded in seven of nine trials for PS 80, while accelerated surfactant adsorption in the presence of rFVIII was recorded in only two of nine trials for poloxamer 188. Experimental uncertainty was generally large, such that the accelerated adsorption recorded for PS 80 in the presence of rFVIII was statistically significant in only three of the seven instances at a 95% confidence level (50 mg/L PS 80 at 40 C and 150 mg/L PS 80 at 4 and 20 C). The two instances of accelerated adsorption recorded for poloxamer 188 in the presence of rFVIII were not statistically significant. Analysis of the rate data The initial rates of surface tension depression recorded for each surfactant were used to determine a rate constant (km) and reaction order (n) at each temperature (T), i.e. initial rate¼km C n where C is surfactant concentration. The activation energy, Ea, for adsorption in each case was derived from the slope of a plot of ln km versus T1 , after Arrhenius expansion of the rate constant, i.e. Ea 1 þ lnð AÞ lnðkm Þ¼ R T where R is the gas constant and A is the Arrhenius preexponential factor (Figure 4). The activation energies determined in this way for adsorption of PS 80 with and without rFVIII were 6.8 ± 6.1 kJ/mol and 8.4 ± 1.7 kJ/mol, respectively. The activation energies for adsorption of poloxamer 188 with and without rFVIII were determined to be 29.4 ± 15.1 kJ/mol and 18.6 ± 1.1 kJ/mol, respectively (errors are based on the sample variation in slopes within the 95% confidence interval in each case). The presence of protein reduced the activation energy for adsorption of PS 80, but did not reduce the activation energy for adsorption of poloxamer 188.
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Pharm Dev Technol, Early Online: 1–5
Figure 3. Initial rate of surface tension depression of PS 80 (left) and poloxamer 188 (right) with and without rFVIII. Trials conducted with rFVIII represented as blank fill, trials without rFVIII represented as colored fill. Error bars represent 95% confidence levels.
Discussion The decrease in Ea for PS 80 adsorption in the presence of rFVIII is consistent with the mechanism depicted in the middle panel of Figure 1, while the absence of such a decrease in Ea for poloxamer 188 adsorption is consistent with the mechanism depicted in the bottom panel of Figure 1. Qualitatively, these results support the notion that PS 80 has a stronger affinity for the air–water interface than for hydrophobic regions on rFVIII, while poloxamer 188 preferentially adsorbs to rFVIII rather than the air-water interface. Additional justification for this notion lies in the fact that the presence of salt is known to lower the critical aggregation concentration of triblock polymers containing PEO or other polyethers8, and the surface at an air–water interface is depleted of salts relative to the bulk solution9. Thus our results provide a rationale for results recorded by Joshi et al. and Kim et al. showing poloxamer 188 to more effectively stabilize rFVIII against aggregation in solution, as well as adsorption loss, relative to PS 80. Our analysis of rate data is based on an equilibrium between the intermediate species and the products in the reaction schemes depicted in the middle and bottom panels of Figure 1, i.e. based on conversion of reactants to the structural intermediate being rate-limiting. Schram & Hall10 carried out this kind of analysis in study of the effect of selected hydrophobic proteins on pulmonary surfactant function, as did Kim et al.7 in study of the effect of lysozyme and a recombinant protein drug on polysorbate and poloxamer adsorption. It is worth noting they also considered the
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Figure 4. Arrhenius plots for poloxamer 188 and PS 80 with and without rFVIII. Activation energy of surfactant adsorption to the interface is calculated using the slope (¼Ea/R). Error bars represent 95% confidence levels.
alternative case, assuming equilibrium between reactants and the structural intermediate, in which case the second step in each scheme (conversion of structural intermediate to products) is the rate-limiting step. In this case, known as ‘‘transition state theory’’11, the rate constant is independent of the nature of the structural intermediate and is defined absolutely as km ¼constant
K b T DG=RT e h
where kb and h are Boltzmann’s and Planck’s constants, respectively, and DG is the free energy of transition. Since DG ¼ DH T DS, the temperature effect on km is well defined and given by ln km DH 1 ln kb DS þconstant þ ¼ þ R T R T h The slope and intercept of plots of ln km/T versus 1/T would therefore provide estimates of the enthalpy (DH) and entropy (DS) of the transition, and DG can thus be calculated at any desired temperature. Invoking transition state theory to describe the temperature effect on protein-mediated acceleration of surfactant adsorption, Schram & Hall as well as Kim et al. found any reduction in DG observed upon addition of protein was owing entirely to a reduction in DH, suggesting that protein is accelerating the adsorption of the surfactants not by reducing the entropic barrier faced by the surfactant in moving from the aggregate to the interface, but by disrupting the surfactant self-association.
DOI: 10.3109/10837450.2014.987297
Stabilization of a human recombinant factor VIII by poloxamer 188
Conclusions Our results are consistent with those of earlier reports suggesting that characterization of protein-mediated acceleration of surfactant adsorption could be used to quantitatively compare different surfactants in relation to their favored mechanism for imparting protein stability and to guide development of surfactant chemistries optimized for protein stabilization under specific conditions. This kind of approach to surfactant selection and design for reduction of aggregation and adsorption loss does not currently exist, and we provide in this study a rationale for hypotheses to drive further discovery and understanding in this important area.
Acknowledgements We thank Dr Omkar Joshi of Bayer Healthcare, LLC, Berkeley, CA, for providing the rFVIII.
Downloaded by [University of Lethbridge] at 13:41 08 November 2015
Declaration of interest We report no declaration of interest.
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