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Freeze-drying of HESylated IFNα-2b: Effect of HESylation on storage stability in comparison to PEGylation
International Journal of Pharmaceutics 495 (2015) 608–611
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Rapid communication
Freeze-drying of HESylated IFNa-2b: Effect of HESylation on storage stability in comparison to PEGylation Robert Liebnera,* , Sarah Bergmannb , Thomas Heyb , Gerhard Wintera , Ahmed Besheera a Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Butenandtstr. 5-8, 81377 Munich, Germany b Innovation Centre Complex Formulations, Fresenius Kabi Deutschland GmbH, Pfingstweide 53, 61169 Friedberg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 July 2015 Received in revised form 14 September 2015 Accepted 15 September 2015 Available online 24 September 2015
A comparison of lyophilized PEGylated and HESylated IFNa was carried out to investigate the influence of protein conjugation, lyoprotectants as well as storage temperature on protein stability. Results show that PEG tends to crystallize during freeze-drying, reducing protein stability upon storage. In contrast, HESylation1 drastically improved the stability over PEGylation by remaining totally amorphous during lyophilization, with and without lyoprotectants while providing a high glass transition temperature of the freeze-dried cakes. ã 2015 Elsevier B.V. All rights reserved.
Keywords: PEGylation HESylation Freeze-drying Phase separation Interferon a-2b Crystallization
Lyophilization of PEGylated proteins and peptides is challenging because PEG, either used as a bulking agent or chemically conjugated, tends to give rise to amorphous phase separation during lyophilization, which is a precursor for crystallization (Bhatnagar et al., 2010a Izutsu et al., 1996). A consequence is a lower storage stability if crystallization is not suppressed by amorphous lyoprotectants (Bhatnagar et al., 2010a,b; Izutsu et al., 1996; Kreilgaard et al., 1998). Amorphous disaccharides like sucrose are frequently used to stabilize proteins during lyophilization and subsequent storage in the dried state (Allison et al., 1999) and are reported to decrease crystallization (Wang, 2000). Meanwhile, many alternative methods have been developed to increase the serum half-life of small sized proteins and peptides and which in addition overcome the drawbacks of PEG (Besheer et al., 2013; McDonnell et al., 2013; Schellekens et al., 2013), including HESylation. The latter has proven its ability to prolong circulation time (Hey et al., 2012; Liebner et al., 2014) and increase the stability of proteins, even at high concentrations (Liebner et al., 2014; Liebner et al., 2015). HES has long been used as a bulking agent and stabilizer during lyophilization based on several facts. HES in solution is characterized by high glass-transition temperatures of the maximally freeze-concentrated matrix of the frozen
* Corresponding author. Fax: +49 89 2180 77020. E-mail address: [email protected] (R. Liebner). http://dx.doi.org/10.1016/j.ijpharm.2015.09.031 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
solution (known as Tg prime or Tg’). During drying process HES provides excellent glass-forming properties with high glass transition temperatures (Tg value) of the solid cakes. (Carpenter et al., 1997). The use of HES as bulking agent and/or lyoprotectant can increase the storage stability in combination with disaccharides, even at higher temperatures (Garzon-Rodriguez et al., 2004). Here, we compare how the covalent attachment with either polyethylene glycol (known as PEGylation) or hydroxyethyl starch (known as HESylation) influence the stability of lyophilized interferon a-2b (IFNa). Therefore, the native IFNa was PEGylated and HESylated by reductive amination in a regioselective manner followed by a stability study of the lyophilized product (see Supplementary material). Initially, both conjugates were characterized to confirm successful conjugation and ensure sufficient purity. SDS-PAGE, RP-HPLC and SEC conducted with conjugate samples before/after chromatographic purification reveal information on the conjugate purity (Fig. 1) and showed that multi-conjugated and unmodified IFNa (low molecular weight band at 15–20 kDa in the SDS-PAGE image) is no longer detectable in the purified fractions (Fig. 1, lane 5: purified monoHESylated IFNa and lane 6: purified monoPEGylated IFNa). Meanwhile, AF4 analysis equipped with MALLS, RI and UV-detectors shows that, for both conjugates, the molecular weight calculated for the polymer fraction is almost identical to the value determined before conjugation, clearly suggesting a 1:1 stoichiometry (see Table 1). In addition, only a single major
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611
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Fig. 1. SDS-PAGE: HES-IFNa before (lane 1) and after (lane 5) purification, PEG-IFNa before (lane 2) and after (lane 6) purification, native IFNa (lane 4), Protein standard (lane M1, M2), (A + C) SEC and RP-HPLC of PEG-IFNa, respectively (B + D) SEC and RP-HPLC of HES-IFNa, respectively.
Table 1 Weight average molar mass (Mw) and polydispersity index (PDI) for the activated polymers, native protein and the conjugates. Sample
IFNa PEG-IFNa HES-IFNa Activated PEG Activated HES a b
Hydrodynamic radius [nm]b
PDI
Conjugate
Modifier/polymer
Protein
Polymer/protein ratio
b
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Modifier in conjugate/modifier before conjugationa
n.a. 68.4 101.2 n.a.
n.a. 65.8 85.8 n.a.
n.a. 47.9 82.0 42.8
n.a. 46.1 61.4 40.4
19.8 20.5 19.2 n.a.
19.2 19.7 18.9 n.a.
n.a. 1.14 1.01 n.a.
n.a.
81.0
60.5
n.a.
n.a.
n.a.
2.1 6.1 5.9 4.7
0.26 0.21 0.54 0.23
4.7
0.55 n.a.
Determined by AF4-MALLS. Determined by DLS.
conjugation site for the polymers was identified at the N-terminus of the protein by tryptic peptide mapping (data not shown). Finally, DLS confirmed a comparable hydrodynamic radius for both conjugates (see Table 1). In terms of lyophilization, the general goal is maintaining the collapse temperature (Tc) as high as possible (Carpenter et al., 1997), which will have a positive impact on the economy of the lyophilization cycle (Carpenter et al., 2002), since an increase of 1 C in product temperature during lyophilization lowers the time for primary drying by about 13% (Tang and Pikal, 2004). Additionally, Tg’ is in a direct relation to the maximally allowed product temperature, which has to be several degrees below Tc to maintain an acceptable appearance of the dried cake (Tang and Pikal, 2004). Basically, the hydroxyethyl starch provides an extraordinary high collapse temperature of the frozen solution in a range of 12 to 17 C depending on the molecular weight, the degree of substitution and the HES to disaccharide ratio (Chen et al., 2002; Sun et al., 2004; Wang, 2000). In that relation, Tg’ was evaluated depending on disaccharide to protein ratio (Table 2). Our results suggested only a slight increase in the Tg’ value for HESylated IFNa. The total amount of HES in the formulation of 1 mg/mL protein concentration is rather low and has almost no effect on Tg’ and process settings.
Table 2 Thermal analysis of the frozen formulations before lyophilization, * not detectable. Sample
Protein conc. [%m/v]
Sucrose conc. [%m/v]
Tg’ [ C]
PEGylated IFNa
0.1 0.1 0.1 0.1 0.1 0.1
0 2.5 5 0 2.5 5
*
HESylated IFNa
35 33 * 31 33
Storage stability correlates well with Tg of the dried cakes and should be at least 20 C above the ambient storage temperature (Wang, 2000). Tg measurements for the different formulations tested indicate that the Tg values of 60 C up to 110 C in the presence of <1% residual moisture were obtained (Fig. 2). Immediately after freeze-drying the native and PEGylated IFNa showed a glass transition temperature in a range between 69 and 74 C for the sucrose based formulations, which remained more or less constant during storage. In contrast, the Tg of HES-IFNa exceeded 110 C for the sucrose free formulation. The addition of 2.5% or 5% sucrose dilutes the effect of the Tg modifying effect of the HES part, leading to Tgs of 75 C and 65 C, respectively.
610
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611
Fig. 2. DSC measurement (A): black bars: Tg after freeze-drying; grey-dashed bars: after 3 months storage at 4 C, white—dashed bars: after 3 months storage at 40 C; Enthalpy per gram of crystallization peak (B): black bars represent the initial value after freeze-drying; the grey-dashed ones after 3 months storage at 4 C and the whitedashed ones after 3 months storage at 40 C.
Fig. 3. Monomer content and levels of high—molecular weight species (HMW) after 3 months at 4 or 40 C.
Additionally, no crystallization can be observed for HES, which is known to be amorphous (Carpenter et al., 2002; Garzon-Rodriguez et al., 2004). For PEG, crystallization was inhibited in the presence of 5% m/v sucrose, but not with 2.5% or in the absence of sucrose (Fig. 2B), which is in agreement with earlier results for lyophilized PEG-hGH (Bhatnagar et al., 2011). Results of storage stability show a considerable effect for storage temperature. While storage at 4 C showed no effect on stability, storage at 40 C show that the formulations with crystallized PEG (Fig. 2B) demonstrate a higher tendency for protein aggregation (Fig. 3), leading to aggregation of 6.9% in the absence of sucrose and 5.5% with 2.5% of sucrose after 3 months of storage. Increasing the concentration of sucrose to 5% significantly decreased the level of soluble aggregates to 1.3%. HES-IFNa showed neither a dramatic decrease in monomer recovery nor soluble aggregates were formed, even at 40 C. In conclusion, the presented study reveals that HESylation can offer a number of advantages over PEGylation in terms of the development of freeze-dried formulations of polymer-proteinconjugates, such as the higher glass transition temperature, as well as the amorphous nature, which maintains storage stability even in the presence of no or low concentration of lyoprotectants. The latter case can be used for up-concentration of protein solutions or to allow a high protein load, which would be attractive for further
manufacturing steps, e.g. bulk drying of drug substance or preparation of drug delivery systems. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.09.031. References Allison, S.D., Chang, B., Randolph, T.W., Carpenter, J.F., 1999. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch. Biochem. Biophys. 365, 289–298. Besheer, A., Liebner, R., Meyer, M., Winter, G., 2013. Challenges for PEGylated proteins and alternative half-life extension technologies based on biodegradable polymers, tailored polymer architectures for pharmaceutical and biomedical applications. Am. Chem. Soc. 215–233. Bhatnagar, B.S., Martin, S.M., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2010a. Investigation of PEG crystallization in frozen PEG – sucrose – water solutions: I. Characterization of the nonequilibrium behavior during freezethawing. J. Pharm. Sci. 99, 2609–2619. Bhatnagar, B.S., Martin, S.M., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2010b. Investigation of PEG crystallization in frozen PEG – sucrose – water solutions: II. Characterization of the equilibrium behavior during freezethawing. J. Pharm. Sci. 99, 4510–4524. Bhatnagar, B.S., Martin, S.W.H., Hodge, T.S., Das, T.K., Joseph, L., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2011. Investigation of PEG crystallization in
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611 frozen and freeze-dried PEGylated recombinant human growth hormone– sucrose systems: implications on storage stability. J. Pharm. Sci. 100, 3062– 3075. Carpenter, J., Chang, B., Garzon-Rodriguez, W., Randolph, T., 2002. Rational design of stable lyophilized protein formulations: theory and practice. In: Carpenter, J., Manning, M. (Eds.), Rational Design of Stable Protein Formulations. Springer, US, pp. 109–133. Carpenter, J., Pikal, M., Chang, B., Randolph, T., 1997. Rational design of stable lyophilized protein formulations: some practical advice. Pharm. Res. 14, 969– 975. Chen, T., Bhowmick, S., Sputtek, A., Fowler, A., Toner, M., 2002. The glass transition temperature of mixtures of trehalose and hydroxyethyl starch. Cryobiology 44, 301–306. Garzon-Rodriguez, W., Koval, R.L., Chongprasert, S., Krishnan, S., Randolph, T.W., Warne, N.W., Carpenter, J.F., 2004. Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethyl starch mixtures. J. Pharm. Sci. 93, 684–696. Hey, T., Knoller, H., Vorstheim, P., 2012. Half-Life Extension Through HESylation1, Therapeutic Proteins. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 117–140. Izutsu, K.-i., Yoshioka, S., Kojima, S., Randolph, T., Carpenter, J., 1996. Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers. Pharm. Res. 13, 1393–1400.
611
Kreilgaard, L., Frokjaer, S., Flink, J.M., Randolph, T.W., Carpenter, J.F., 1998. Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid. Arch. Biochem. Biophys. 360, 121–134. Liebner, R., Mathaes, R., Meyer, M., Hey, T., Winter, G., Besheer, A., 2014. Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra. Eur. J. Pharm. Biopharm. 87, 378–385. Liebner, R., Meyer, M., Hey, T., Winter, G., Besheer, A., 2015. Head to head comparison of the formulation and stability of concentrated solutions of HESylated versus PEGylated anakinra. J. Pharm. Sci. 104, 515–526. McDonnell, T., Ioannou, Y., Rahman, A., 2013. PEGylated drugs in rheumatology— why develop them and do they work? Rheumatology.. Schellekens, H., Hennink, W., Brinks, V., 2013. The immunogenicity of polyethylene glycol: facts and fiction. Pharm. Res. 30, 1729–1734. Sun, W.Q., Wagner, C.T., Connor, J., 2004. The glass transition behaviors of hydroxyethyl starch solutions. Cell Preserv. Technol. 2, 55–65. Tang, X., Pikal, M., 2004. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm. Res. 21, 191–200. Wang, W., 2000. Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm. 203, 1–60.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Rapid communication
Freeze-drying of HESylated IFNa-2b: Effect of HESylation on storage stability in comparison to PEGylation Robert Liebnera,* , Sarah Bergmannb , Thomas Heyb , Gerhard Wintera , Ahmed Besheera a Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximillians-University Munich, Butenandtstr. 5-8, 81377 Munich, Germany b Innovation Centre Complex Formulations, Fresenius Kabi Deutschland GmbH, Pfingstweide 53, 61169 Friedberg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 July 2015 Received in revised form 14 September 2015 Accepted 15 September 2015 Available online 24 September 2015
A comparison of lyophilized PEGylated and HESylated IFNa was carried out to investigate the influence of protein conjugation, lyoprotectants as well as storage temperature on protein stability. Results show that PEG tends to crystallize during freeze-drying, reducing protein stability upon storage. In contrast, HESylation1 drastically improved the stability over PEGylation by remaining totally amorphous during lyophilization, with and without lyoprotectants while providing a high glass transition temperature of the freeze-dried cakes. ã 2015 Elsevier B.V. All rights reserved.
Keywords: PEGylation HESylation Freeze-drying Phase separation Interferon a-2b Crystallization
Lyophilization of PEGylated proteins and peptides is challenging because PEG, either used as a bulking agent or chemically conjugated, tends to give rise to amorphous phase separation during lyophilization, which is a precursor for crystallization (Bhatnagar et al., 2010a Izutsu et al., 1996). A consequence is a lower storage stability if crystallization is not suppressed by amorphous lyoprotectants (Bhatnagar et al., 2010a,b; Izutsu et al., 1996; Kreilgaard et al., 1998). Amorphous disaccharides like sucrose are frequently used to stabilize proteins during lyophilization and subsequent storage in the dried state (Allison et al., 1999) and are reported to decrease crystallization (Wang, 2000). Meanwhile, many alternative methods have been developed to increase the serum half-life of small sized proteins and peptides and which in addition overcome the drawbacks of PEG (Besheer et al., 2013; McDonnell et al., 2013; Schellekens et al., 2013), including HESylation. The latter has proven its ability to prolong circulation time (Hey et al., 2012; Liebner et al., 2014) and increase the stability of proteins, even at high concentrations (Liebner et al., 2014; Liebner et al., 2015). HES has long been used as a bulking agent and stabilizer during lyophilization based on several facts. HES in solution is characterized by high glass-transition temperatures of the maximally freeze-concentrated matrix of the frozen
* Corresponding author. Fax: +49 89 2180 77020. E-mail address: [email protected] (R. Liebner). http://dx.doi.org/10.1016/j.ijpharm.2015.09.031 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
solution (known as Tg prime or Tg’). During drying process HES provides excellent glass-forming properties with high glass transition temperatures (Tg value) of the solid cakes. (Carpenter et al., 1997). The use of HES as bulking agent and/or lyoprotectant can increase the storage stability in combination with disaccharides, even at higher temperatures (Garzon-Rodriguez et al., 2004). Here, we compare how the covalent attachment with either polyethylene glycol (known as PEGylation) or hydroxyethyl starch (known as HESylation) influence the stability of lyophilized interferon a-2b (IFNa). Therefore, the native IFNa was PEGylated and HESylated by reductive amination in a regioselective manner followed by a stability study of the lyophilized product (see Supplementary material). Initially, both conjugates were characterized to confirm successful conjugation and ensure sufficient purity. SDS-PAGE, RP-HPLC and SEC conducted with conjugate samples before/after chromatographic purification reveal information on the conjugate purity (Fig. 1) and showed that multi-conjugated and unmodified IFNa (low molecular weight band at 15–20 kDa in the SDS-PAGE image) is no longer detectable in the purified fractions (Fig. 1, lane 5: purified monoHESylated IFNa and lane 6: purified monoPEGylated IFNa). Meanwhile, AF4 analysis equipped with MALLS, RI and UV-detectors shows that, for both conjugates, the molecular weight calculated for the polymer fraction is almost identical to the value determined before conjugation, clearly suggesting a 1:1 stoichiometry (see Table 1). In addition, only a single major
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611
609
Fig. 1. SDS-PAGE: HES-IFNa before (lane 1) and after (lane 5) purification, PEG-IFNa before (lane 2) and after (lane 6) purification, native IFNa (lane 4), Protein standard (lane M1, M2), (A + C) SEC and RP-HPLC of PEG-IFNa, respectively (B + D) SEC and RP-HPLC of HES-IFNa, respectively.
Table 1 Weight average molar mass (Mw) and polydispersity index (PDI) for the activated polymers, native protein and the conjugates. Sample
IFNa PEG-IFNa HES-IFNa Activated PEG Activated HES a b
Hydrodynamic radius [nm]b
PDI
Conjugate
Modifier/polymer
Protein
Polymer/protein ratio
b
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Mw by RI/ MALLS [kDa]a
Mn by RI/ MALLS [kDa]a
Modifier in conjugate/modifier before conjugationa
n.a. 68.4 101.2 n.a.
n.a. 65.8 85.8 n.a.
n.a. 47.9 82.0 42.8
n.a. 46.1 61.4 40.4
19.8 20.5 19.2 n.a.
19.2 19.7 18.9 n.a.
n.a. 1.14 1.01 n.a.
n.a.
81.0
60.5
n.a.
n.a.
n.a.
2.1 6.1 5.9 4.7
0.26 0.21 0.54 0.23
4.7
0.55 n.a.
Determined by AF4-MALLS. Determined by DLS.
conjugation site for the polymers was identified at the N-terminus of the protein by tryptic peptide mapping (data not shown). Finally, DLS confirmed a comparable hydrodynamic radius for both conjugates (see Table 1). In terms of lyophilization, the general goal is maintaining the collapse temperature (Tc) as high as possible (Carpenter et al., 1997), which will have a positive impact on the economy of the lyophilization cycle (Carpenter et al., 2002), since an increase of 1 C in product temperature during lyophilization lowers the time for primary drying by about 13% (Tang and Pikal, 2004). Additionally, Tg’ is in a direct relation to the maximally allowed product temperature, which has to be several degrees below Tc to maintain an acceptable appearance of the dried cake (Tang and Pikal, 2004). Basically, the hydroxyethyl starch provides an extraordinary high collapse temperature of the frozen solution in a range of 12 to 17 C depending on the molecular weight, the degree of substitution and the HES to disaccharide ratio (Chen et al., 2002; Sun et al., 2004; Wang, 2000). In that relation, Tg’ was evaluated depending on disaccharide to protein ratio (Table 2). Our results suggested only a slight increase in the Tg’ value for HESylated IFNa. The total amount of HES in the formulation of 1 mg/mL protein concentration is rather low and has almost no effect on Tg’ and process settings.
Table 2 Thermal analysis of the frozen formulations before lyophilization, * not detectable. Sample
Protein conc. [%m/v]
Sucrose conc. [%m/v]
Tg’ [ C]
PEGylated IFNa
0.1 0.1 0.1 0.1 0.1 0.1
0 2.5 5 0 2.5 5
*
HESylated IFNa
35 33 * 31 33
Storage stability correlates well with Tg of the dried cakes and should be at least 20 C above the ambient storage temperature (Wang, 2000). Tg measurements for the different formulations tested indicate that the Tg values of 60 C up to 110 C in the presence of <1% residual moisture were obtained (Fig. 2). Immediately after freeze-drying the native and PEGylated IFNa showed a glass transition temperature in a range between 69 and 74 C for the sucrose based formulations, which remained more or less constant during storage. In contrast, the Tg of HES-IFNa exceeded 110 C for the sucrose free formulation. The addition of 2.5% or 5% sucrose dilutes the effect of the Tg modifying effect of the HES part, leading to Tgs of 75 C and 65 C, respectively.
610
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611
Fig. 2. DSC measurement (A): black bars: Tg after freeze-drying; grey-dashed bars: after 3 months storage at 4 C, white—dashed bars: after 3 months storage at 40 C; Enthalpy per gram of crystallization peak (B): black bars represent the initial value after freeze-drying; the grey-dashed ones after 3 months storage at 4 C and the whitedashed ones after 3 months storage at 40 C.
Fig. 3. Monomer content and levels of high—molecular weight species (HMW) after 3 months at 4 or 40 C.
Additionally, no crystallization can be observed for HES, which is known to be amorphous (Carpenter et al., 2002; Garzon-Rodriguez et al., 2004). For PEG, crystallization was inhibited in the presence of 5% m/v sucrose, but not with 2.5% or in the absence of sucrose (Fig. 2B), which is in agreement with earlier results for lyophilized PEG-hGH (Bhatnagar et al., 2011). Results of storage stability show a considerable effect for storage temperature. While storage at 4 C showed no effect on stability, storage at 40 C show that the formulations with crystallized PEG (Fig. 2B) demonstrate a higher tendency for protein aggregation (Fig. 3), leading to aggregation of 6.9% in the absence of sucrose and 5.5% with 2.5% of sucrose after 3 months of storage. Increasing the concentration of sucrose to 5% significantly decreased the level of soluble aggregates to 1.3%. HES-IFNa showed neither a dramatic decrease in monomer recovery nor soluble aggregates were formed, even at 40 C. In conclusion, the presented study reveals that HESylation can offer a number of advantages over PEGylation in terms of the development of freeze-dried formulations of polymer-proteinconjugates, such as the higher glass transition temperature, as well as the amorphous nature, which maintains storage stability even in the presence of no or low concentration of lyoprotectants. The latter case can be used for up-concentration of protein solutions or to allow a high protein load, which would be attractive for further
manufacturing steps, e.g. bulk drying of drug substance or preparation of drug delivery systems. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.09.031. References Allison, S.D., Chang, B., Randolph, T.W., Carpenter, J.F., 1999. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch. Biochem. Biophys. 365, 289–298. Besheer, A., Liebner, R., Meyer, M., Winter, G., 2013. Challenges for PEGylated proteins and alternative half-life extension technologies based on biodegradable polymers, tailored polymer architectures for pharmaceutical and biomedical applications. Am. Chem. Soc. 215–233. Bhatnagar, B.S., Martin, S.M., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2010a. Investigation of PEG crystallization in frozen PEG – sucrose – water solutions: I. Characterization of the nonequilibrium behavior during freezethawing. J. Pharm. Sci. 99, 2609–2619. Bhatnagar, B.S., Martin, S.M., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2010b. Investigation of PEG crystallization in frozen PEG – sucrose – water solutions: II. Characterization of the equilibrium behavior during freezethawing. J. Pharm. Sci. 99, 4510–4524. Bhatnagar, B.S., Martin, S.W.H., Hodge, T.S., Das, T.K., Joseph, L., Teagarden, D.L., Shalaev, E.Y., Suryanarayanan, R., 2011. Investigation of PEG crystallization in
R. Liebner et al. / International Journal of Pharmaceutics 495 (2015) 608–611 frozen and freeze-dried PEGylated recombinant human growth hormone– sucrose systems: implications on storage stability. J. Pharm. Sci. 100, 3062– 3075. Carpenter, J., Chang, B., Garzon-Rodriguez, W., Randolph, T., 2002. Rational design of stable lyophilized protein formulations: theory and practice. In: Carpenter, J., Manning, M. (Eds.), Rational Design of Stable Protein Formulations. Springer, US, pp. 109–133. Carpenter, J., Pikal, M., Chang, B., Randolph, T., 1997. Rational design of stable lyophilized protein formulations: some practical advice. Pharm. Res. 14, 969– 975. Chen, T., Bhowmick, S., Sputtek, A., Fowler, A., Toner, M., 2002. The glass transition temperature of mixtures of trehalose and hydroxyethyl starch. Cryobiology 44, 301–306. Garzon-Rodriguez, W., Koval, R.L., Chongprasert, S., Krishnan, S., Randolph, T.W., Warne, N.W., Carpenter, J.F., 2004. Optimizing storage stability of lyophilized recombinant human interleukin-11 with disaccharide/hydroxyethyl starch mixtures. J. Pharm. Sci. 93, 684–696. Hey, T., Knoller, H., Vorstheim, P., 2012. Half-Life Extension Through HESylation1, Therapeutic Proteins. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 117–140. Izutsu, K.-i., Yoshioka, S., Kojima, S., Randolph, T., Carpenter, J., 1996. Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers. Pharm. Res. 13, 1393–1400.
611
Kreilgaard, L., Frokjaer, S., Flink, J.M., Randolph, T.W., Carpenter, J.F., 1998. Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid. Arch. Biochem. Biophys. 360, 121–134. Liebner, R., Mathaes, R., Meyer, M., Hey, T., Winter, G., Besheer, A., 2014. Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra. Eur. J. Pharm. Biopharm. 87, 378–385. Liebner, R., Meyer, M., Hey, T., Winter, G., Besheer, A., 2015. Head to head comparison of the formulation and stability of concentrated solutions of HESylated versus PEGylated anakinra. J. Pharm. Sci. 104, 515–526. McDonnell, T., Ioannou, Y., Rahman, A., 2013. PEGylated drugs in rheumatology— why develop them and do they work? Rheumatology.. Schellekens, H., Hennink, W., Brinks, V., 2013. The immunogenicity of polyethylene glycol: facts and fiction. Pharm. Res. 30, 1729–1734. Sun, W.Q., Wagner, C.T., Connor, J., 2004. The glass transition behaviors of hydroxyethyl starch solutions. Cell Preserv. Technol. 2, 55–65. Tang, X., Pikal, M., 2004. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm. Res. 21, 191–200. Wang, W., 2000. Lyophilization and development of solid protein pharmaceuticals. Int. J. Pharm. 203, 1–60.