Solid State Characterization of Spray-Dried Powders of Recombinant Human Deoxyribonuclease (RhDNase) † HAK-KIM CHAN* ,‡ AND IGOR GONDA § Contribution from the Department of Pharmacy, A15, University of Sydney, NSW 2006, Australia, and Aradigm Corporation, Hayward, California 94545. Received October 11, 1995. Abstract 0 rhDNase is a recombinant human protein approved as an aqueous solution for human use by inhalation. To study the feasibility of preparation of dry powders for inhalation, spray-dried powders of pure rhDNase and co-spray-dried mixtures of rhDNase with an excipient approved for inhalation products, lactose, were prepared. Both types of powders were initially amorphous. The lactose, however, was found to crystallize after exposure to a humid environment. The crystals in the powder were identified as the R-monohydrate polymorph of lactose by hot-stage optical and scanning electron microscopy, differential scanning calorimetry and thermo- gravimetry, FTIR spectroscopy, and X-ray powder diffraction. Moisture sorption isotherms indicated that crystallization occurred at high relative humidities (70-85%), depending on the temperature of the environment (5-40 °C). The practical implications for the manufacturing and storage of protein powders for inhalation are discussed. Introduction Recombinant human deoxyribonuclease (rhDNase, R-dor- nase) is the first recombinant protein approved for human therapy by inhalation. 1,2 It is available as an aqueous solution for nebulization. 3-5 The relatively low efficiency 4,5 and lack of convenience associated with the use of nebu- lizers prompted us to investigate alternative inhalation systems. The work reported here is related to our efforts to prepare a formulation suitable for delivery of rhDNase with dry powder inhalers (DPIs). Relatively little has been published thus far on systematic studies of respirable protein powders. This report is likely to be particularly relevant to future work on protein formulations for DPIs since rhDNase represents a wide class of glycosylated hydrophilic proteins that may be advantageously delivered by inhalation for local or systemic effects. 6 Powder inhala- tion delivery systems for small molecules have been gaining wide acceptance, primarily for the treatment of asthma. DPIs require the ability to generate aerosols from powders containing drug particles in the micron size range. 7,8 To minimize degradation of proteins during manufacturing and storage, especially by molecular aggregation, it is necessary to protect the protein to prevent undesirable protein-protein interactions. For example, stable lyoph- ilized protein formulations contain high proportions of excipients, such as sugars, that have the ability to protect the protein against denaturation during freezing as well as to act as a vitreous matrix that slows down intra- and inter-molecular interactions. 9 There is very limited knowl- edge of the inhalation safety of these excipients, and a conservative approach is typically taken when employing some of the few materials approved for inhalation therapy such as lactose. Moreover, lyophilized protein powders do not have the particle size and dispersibility characteristics suitable for inhalation. Therefore, the purpose of the overall project was to investigate the feasibility of the use lactose for preparation of respirable powders of rhDNase by spray-drying rather than freeze-drying. A better un- derstanding of the solid-state properties of spray-dried lactose-rhDNase powders was an essential component in the research toward stable respirable formulations that could lead to a safe and efficacious protein dosage form. Lactose is a naturally occurring disaccharide that con- sists of one galactose and one glucose unit. It exhibits mutarotation in two isomeric, R and , forms, resulting in an equilibrium mixture of R- and -lactose ranging from 1 part R to 1.65 parts  at 0 °C to 1 part R to 1.33 parts  at 100 °C. 10 Solid lactose can exist in various forms: 10-12 R-lactose monohydrate (nonhygroscopic, prepared by crys- tallization from an aqueous solution at temperature <93 °C); unstable anhydrous R-lactose (hygroscopic, prepared by heating R-lactose monohydrate at 100-130 °C); stable anhydrous R-lactose (nonhygroscopic, prepared by heating R-lactose monohydrate at >130 °C); anhydrous -lactose (nonhygroscopic, prepared by crystallization from an aque- ous solution at temperature >93 °C); and amorphous lactose (hygroscopic, prepared by rapid drying of a lactose solution, e.g. by spray-drying). It has been shown that it is feasible to obtain dry powders of undenatured proteins by spray-drying. 13 We have recently prepared spray-dried rhDNase containing lactose for aerosol delivery. 14-16 A significant loss in the dispersibility of the powders was found after their storage at high relative humidities. 15 An initial sorption isotherm study of the spray-dried rhDNase-lactose powder showed a discontinuity at high relative humidity (RH ∼80%) during moisture uptake. Further studies using microscopy, ther- mal analysis, FTIR spectroscopy, and X-ray powder dif- fraction were therefore carried out to improve our under- standing of the nature of these changes as they throw much light on the feasibility of preparation of stable formulations of not just rhDNase but other proteins as well. Materials and Methods Spray-dried powders were prepared from rhDNase solutions containing different concentrations of lactose in a Bu ¨ chi 190 Mini Spray Dryer at inlet and outlet temperatures of 90 and 55 °C, respectively. The solutions were fed at a rate of 5 mL/min. The composition of the powders initially equilibrated at room temper- ature and RH ∼45%, and subsequently stored in refrigerated capped vials, was (percentage by weight): (1) rhDNase 91.7%, moisture 8.3% and (2) rhDNase 53.5%, lactose 40.4%, and moisture 6.1% (the rhDNase content was measured by UV spetrophotometry † Presented in part in the western regional AAPS meeting, San Jose, 1995. * Corresponding author. Phone: 011-61-2-9351-3054. Fax: 011-61- 2-9351-4391. E-mail: kimc@pharm.usyd.edu.au. ‡ University of Sydney. § Aradigm Corp. © 1998, American Chemical Society and S0022-3549(95)00429-1 CCC: $15.00 Journal of Pharmaceutical Sciences / 647 American Pharmaceutical Association Vol. 87, No. 5, May 1998 Published on Web 04/01/1998