Effects of surfactant

From the formulations investigated in this study, myoglobin formulated with trehalose in 25 mM histidine at pH 6.2 (formulation MT6) showed the best results in terms of maintaining both the pH and myoglobin integrity. However, despite the observation that the reconstituted pH was close to the original pH, this formulation also exhibited some aggregation after CO2 spray drying. It was previously

reported that myoglobin aggregation most likely occurs during scCO2

spray drying due to exposure of the protein at the interface between the atomized myoglobin droplets and the pressurized CO2 [5]. Based on

these results, it was hypothesized that adding a surfactant to this formulation will act as a barrier between the water phase and the CO2,

thus limiting the exposure of the myoglobin to the CO2. By doing so, the

myoglobin is expected to remain in a hydrophilic environment, thereby inhibiting the protein unfolding that leads to aggregation. In this study, polysorbate 80 was included in the 25 mM histidine formulation (MT6) in order to prevent surface-induced protein aggregation (formulations MT7-9).

In biopharmaceutics, nonionic surfactants such as polysorbate 20 and polysorbate 80 are generally used to stabilize protein formulations in a solution [37]. However, in this current study, the addition of polysorbate 80 resulted in almost no improvement in the myoglobin monomer recovery when compared to the formulation without surfactant (Table 2). Moreover, even at the highest polysorbate concentration tested there was neither a reduction in the total sub- visible particle concentration (Fig. 5) nor an increase in the total

myoglobin content after spray drying (Table 1). From these results, it can be concluded that the surfactant is ineffective under these conditions, which may be related to the surfactant concentration, the choice of buffer excipients and the scCO2 spray drying conditions. It has previously

been observed that nonionic biocompatible surfactants, like polysorbate 80, can be used to prepare CO2/water and water/CO2

nano- and microemulsions, but require relatively high surfactant

concentrations (typically 0.5-1.0%wt. exceeding polysorbate

concentrations up to max. 0.1%wt typically used in protein formulations), depending on the pressure and temperature, stirring rate, water and salt concentrations [38].

5. Conclusion

We investigated the role of the formulation excipients on stabilizing myoglobin during scCO2 spray drying. In particular, the

excipients used in the formulation should maintain the heme and overall structure of myoglobin while minimizing aggregation. While trehalose helped to retain the heme within the binding pocket, it was unable to prevent the acidification of the myoglobin solutions during spraying, which resulted in some heme loss and myoglobin aggregation. Heme stabilization in myoglobin was achieved when the pH of protein formulations was maintained at pH 6.2, by using a high concentration of phosphate buffer or a low concentration of citrate or histidine buffer. Furthermore, the phosphate and histidine buffers, but not the citrate buffer, reduced myoglobin aggregation. In the absence of classical buffers, myoglobin presented self-buffering and self-stabilizing properties at high concentration. The addition of polysorbate 80 to the myoglobin formulation did not have a significant effect on reducing myoglobin aggregation. From this study, it can be concluded that excipients are required to protect the protein against process-induced damage during scCO2 spray drying.

References

[1] N. Jovanović, A. Bouchard, G. W. Hofland, G. Witkamp, D. J. A. Crommelin, W.

Jiskoot, Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying, Eur J Pharm Sci, 27 (2006) 336-345. [2] N. Jovanović, A. Bouchard, G. W. Hofland, D. J. Crommelin, W. Jiskoot,

Stabilization of IgG by supercritical fluid drying: optimization of formulation and process parameters, Eur J Pharm Biopharm, 68 (2008) 183-190.

[3] N. Jovanović, A. Bouchard, M. Sutter, M. V. Speybroeck, G. W. Hofland, G. Witkamp, D. J. A. Crommelin, W. Jiskoot, Stable sugar-based protein formulations by supercritical fluid drying, Int J Pharm, 346 (2008) 102-108.

[4] O. Nuchuchua, H. A. Every, G. W. Hofland, W. Jiskoot, Scalable organic solvent

free supercritical fluid spray drying process for producing dry protein formulations, Eur J Pharm Biopharm, 88 (2014) 919-930.

[5] O. Nuchuchua, H.A. Every, G.W. Hofland, W. Jiskoot, Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin, Eur J Pharm Biopharm, 103 (2016) 200-9. [6] A. Bouchard, N. Jovanović, G.W. Hofland, W. Jiskoot, E. Mendes, D.J.A.

Crommelin, G.J. Witkamp, Supercritical fluid drying of carbohydrates: Selection of suitable excipients and process conditions, Eur J Pharm Biopharm, 68 (2008) 781-794.

[7] N. P. Bhatt, K. Patel, R.T. Borchardt, Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone, Pharm Res, 7 (1990) 593-599. [8] C. Oliyai, R.T. Borchardt, Chemical pathways of peptide degradation. IV.

Pathways, kinetics, and mechanism of degradation of an aspartyl residue in a model hexapeptide, Pharm Res, 10 (1993) 95-102.

[9] C. Oliyai, R.T. Borchardt, Chemical pathways of peptide degradation. VI. Effect of the primary sequence on the pathways of degradation of aspartyl residues in model hexapeptides, Pharm Res, 11 (1994) 751-758.

[10] K. Patel, R.T. Borchardt, Chemical pathways of peptide degradation. II. Kinetics of deamidation of an asparaginyl residue in a model hexapeptide, Pharm Res, 7 (1990) 703-711.

[11] D.E. Graham, M.C. Phillips, Proteins at Liquid Interfaces .1. Kinetics of Adsorption and Surface Denaturation, Journal of Colloid and Interface Science, 1979. 70(3): p. 403-414.

[12] Z. Yu, K.P. Johnston, R.O. Williams 3rd, Spray freezing into liquid versus spray-freeze drying: influence of atomization on protein aggregation and biological activity, Eur J Pharm Sci, 27 (2006) 9-18.

[13] S. Ohtake, Y. Kita, T. Arakawa, Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev, 63 (2011) 1053-73.

[14] L. S. Jones, N. B. Bam, T. W. Randolph, Surfactant-stabilized protein formulations: A review of protein-surfactant interactions and novel analytical methodologies (Chapter 12) in Therapeutic Protein and Peptide Formulation and Delivery, 1997, 206–222.

[15] M. Adler, G. Lee, Stability and surface activity of lactate dehydrogenase in spray- dried trehalose. J Pharm Sci, 88 (1999) 199-208.

[16] Y.F. Maa, P.A.T. Nguyen, S.W. Hsu, Spray-drying of air-liquid interface sensitive recombinant human growth hormone, J Pharm Sci, 87 (1998) 152-159.

[17] J.D. Morrisett, J.S.K. David, H.J. Pownall, A.M. Gotto Jr, Interaction of an apolipoprotein (apoLP-alanine) with phosphatidylcholine. Biochemistry, 12 (1973) 1290-1299.

[18] A.R. Karow, S. Bahrenburg, P. Garidel, Buffer Capacity of Biologics-From Buffer Salts to Buffering by Antibodies, Biotechnol Prog, 29 (2013) 480-492.

[19] M. Momenteau, M. RougÉE, B. Loock, Five-Coordinate Iron-Porphyrin as a Model

for the Active Site of Hemoproteins Characterization and Coordinating Properties, Eur J Biochem, 71 (1976) 63-76.

[20] E. Peggion, A. Cosani, M. Terbojevich, Solution properties of synthetic polypeptides. Assignment of the conformation of poly(L-tyrosine) in water and in ethanol-water solutions. Macromolecules, 7 (1974) 453-459.

[21] H. Yamamoto, A. Nakazawa, T Hayakawa, Circular-dichroism studies of

complexes of sequential histidine polypeptides with methyl-orange, In. J Biol Macromolec, 7 (1985) 167-172.

[22] M.C. Hsu, R.W. Woody, The origin of the heme Cotton effects in myoglobin and

hemoglobin, J Am Chem Soc, 93 (1971) 3515-25.

[23] M. Nagai, Y. Nagai, K. Imai, S. Neya, Circular dichroism of hemoglobin and myoglobin, Chirality, 26 (2014) 438-442.

[24] M. S. Hargrove, D. Barrick, J.S. Olson, The association rate constant for heme binding to globin is independent of protein structure, Biochemistry, 35 (1996) 11293-11299.

[25] A. N. Schechter, C. J. Epstein, Spectral studies on the denaturation of myoglobin, J Mol Biol, 35 (1968) 567-589.

[26] T. Arakawa, S.N. Timasheff, Stabilization of protein structure by sugars, Biochemistry, 21 (1982) 6536-6544.

[27] N. K. Jain, I. Roy, Trehalose and protein stability, Curr Protoc Protein Sci, 2010, Chapter 4: p. Unit 4 9.

[28] N. K. Jain, I. Roy, Effect of trehalose on protein structure, Protein Sci, 18 (2009) 24- 36.

[29] A. Pyne, K. Chatterjee, R. Suryanarayanan, Solute crystallization in mannitol- glycine systems--implications on protein stabilization in freeze-dried formulations, J Pharm Sci, 92 (2003) 2272-2283.

[30] M. Robinson, A. L. Hemming, J. A. Regnis, A. G. Wong, D. L. Bailey, G. J. Bautovich, M. King, P. T. Bye, Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis, Thorax, 52 (1997) 900-3.

[31] B. Chen, R. Bautista, K. Yu, G. A. Zapata, M. G. Mulkerrin, Influence of histidine on the stability and physical properties of a fully human antibody in aqueous and solid forms, Pharm Res, 20 (2003) 1952-1960.

[32] J. Y. Zheng, L. J. Janis, Influence of pH, buffer species, and storage temperature on physicochemical stability of a humanized monoclonal antibody LA298, Int J Pharm, 308 (2006) 46-51.

[33] M. G. Cacace, E. M. Landau, J. J. Ramsden, The Hofmeister series: salt and solvent effects on interfacial phenomena, Q Rev Biophys, 30 (1997) 241-277.

[34] T. Osterberg, A. Fatouros, M. Mikaelsson, Development of freeze-dried albumin- free formulation of recombinant factor VIII SQ, Pharm Res, 14 (1997) 892-898. [35] A. Cooper, M. W. Kennedy, Biofoams and natural protein surfactants, Biophys

Chem, 151 (2010) 96-104.

[36] S. J. Shire, Z. Shahrokh, J. Liu, Challenges in the development of high protein concentration formulations, J Pharm Sci, 93 (2004), 1390-1402.

[37] T. W. Randolph, L. S. Jones, Surfactant-protein interactions, Pharm Biotechnol, 13 (2002) 159-175.

[38] D. D. V. Slyke, On the measurement of buffer values and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution, J Biol Chem, 52 (1922) 525.

CHAPTER 7