Effect of Vacuum Drying on Protein-Mannitol Interactions: The Physical State of Mannitol and Protein Structure in the Dried State

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Effect of Vacuum Drying on Protein-Mannitol Interactions: The Physical State of Mannitol and Protein Structure in the Dried State
2004
Vikas K. Sharma1,2 and Devendra S. Kalonia1
1Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269
2Present address: Biotechnology Process Engineering Center, and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
AAPS PharmSciTech 2004; 5 (1) Article 10
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ABSTRACT
The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum dry-ing, to obtain a better understanding of the effect of pro-tein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing ?-lactoglobulin (?Lg):mannitol (1:1-1:15 wt/wt) were vacuum dried at 5?C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray pow-der diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1:5 wt/wt ?Lg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples ex-cept for the 1:1 wt/wt ?Lg:mannitol dried sample. FTIR studies indicated that a small proportion of crystalline man-nitol was present along with the amorphous mannitol in dried samples at lower (less than 1:5 wt/wt) ?Lg:mannitol ratios. The Tg of the dried 1:1 wt/wt ?Lg:mannitol sample was observed at 33.4?C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the ?Lg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1:1 wt/wt in all dried samples. Second-derivative FTIR studies on dried ?Lg and recombi-nant human interferon a-2a samples showed that mannitol affected protein secondary structure to a varying degree de-pending on the overall mannitol content in the dried sample and the type of protein.
Introduction
Recently, there has been an increased level of interest in developing drying technologies in addition to lyophilization to formulate proteins as dry powders. The need has been generated to overcome some of the issues associated with the process of lyophilization that include long processing times (typically 3-5 days), expensive set up and mainte-nance of the lyophilization plants, and, most of all, the insta-bilities incurred upon proteins because of the inherent steps involved during freeze-drying.1,2 The alternative technolo-gies to lyophilization that have been developed recently in-clude spray-drying,3,4 spray-freeze drying,5 bulk crystalliza-tion,6 supercritical fluid technology,7,8 vacuum drying,9 and foam drying.10 Due to the complex structural properties, proteins have a tendency to denature and undergo irreversi-ble aggregation during various processing steps of drying.11-13 The general strategy to stabilize proteins against drying and dehydration stresses is to use sugars such as trehalose or sucrose. The protective action of these sugars is attributed to their water-substituent properties,14-16 which would preserve protein native structure upon loss of water, and/or their ten-dency to form stable glassy matrices,17-19 which would pro-vide a kinetic barrier for protein denaturation or aggregation to occur.
Mannitol is often added in dried protein formulations as the bulking agent as it has the tendency to crystallize rapidly from aqueous solutions. However, the physical state of mannitol depends on the processing steps, and improper control of the drying process can significantly affect the physical properties of mannitol in terms of its crystallization behavior. For example, manipulation of the freezing cycle can result in the formation of either amorphous mannitol or crystalline mannitol depending on the duration and the cool-ing rate during the freezing step of lyophilization.20,21 Stud-ies have been reported on the effect of mannitol on the sta-bility of proteins during freeze-drying,22,23 spray-drying,24 and microsphere preparation.25 It has been shown that, in general, protein stability depended on the physical state of mannitol, and crystalline mannitol was ineffective in pre-serving protein activity. Investigations have shown that mannitol crystallinity is affected by the presence of a coso-lute such as proteins, buffer components, or sugars.20,21 Al-though these investigations indicated that a certain cosolute to mannitol ratio is required for the mannitol to remain amorphous, there has been no detailed systematic study to investigate the effect of the protein:mannitol ratio on the crystallinity of mannitol. In certain instances, for example, a higher protein:mannitol ratio (1:50 wt/wt) has been used to investigate protein-mannitol interactions,23 whereas, in other studies, it has been reported that mannitol crystallinity is inhibited even at a much lower protein:mannitol wt/wt ra-tio.24 It is also important to note that in almost all of the stud-ies performed to investigate protein-mannitol interactions, freeze-drying or spray-drying has been used as the drying process. These processes by themselves have the tendency to affect protein structure and stability because of the inher-ent steps involved such as freezing, presence of ice/water interface (in case of freeze-drying,) or generation of a large air/water interface (in case of spray-drying). Hence, it would be difficult to ascertain whether an alteration in protein sta-bility or structure in the presence of mannitol is due to the effect of the process or the inability of the excipient (ie, mannitol) to preserve protein structure/stability.