GUIDELINE #2: UNDERSTAND THE ROLE OF PARTICLE STRUCTURE AND MORPHOLOGY IN PRODUCT FUNCTION AND STABILITY

3. GUIDELINE #2: UNDERSTAND THE ROLE OF PARTICLE STRUCTURE AND MORPHOLOGY IN PRODUCT FUNCTION AND STABILITY

The size and microstructure of a dry powder formulation can make the difference between an effective and ineffective pro- duct. Particle size or porosity for example can affect dissolution properties of the powder or, as for inhalation powders, their ability to deposit in the appropriate part of the lung for absorption (10). Similarly in biodegradable microspheres, the ability to control size and morphology is critical in obtaining desirable release characteristics and optimal stability.

As noted above, microspheres produced by the process described contain protein particles encapsulated in a polymer matrix. The mechanism of protein release involves first the hydration of the microspheres, followed initially by dissolu- tion and diffusion of protein at or with access via pores to the surface (burst), and finally by release through additional pores and channels created by polymer degradation (11–13). The relative size of the protein particles to the microsphere is an important parameter in controlling the initial release or burst. The particle size of the protein powder can be con- trolled by varying SFD process parameters (14). Protein particles with sizes from several microns down to less than

1 mm are produced by sonicating particles made by SFD with different mass flow ratios (ratio of atomization air =liquid mass flow rates). Figure 3 shows that encapsulating submi- cron protein particles results in a substantially lower initial release than particles much larger than 1 micron (14).

Biodegradable Microspheres for the Sustained Release of Proteins 577

Figure 3 Effects of protein particle size on initial release in vitro. The particle size represents the median protein particle size mea- sured after sonication in a PLG-methylene chloride solution using

a Coulter counter. The initial release represents the percentage of protein released from microspheres within the first 24 hr after incu- bation in a physiological buffer at 37 C.

It is interesting to note how these small protein particles are formed. Spray-freeze drying alone produced particles with a characteristic size of about 10–50 mm, too large to be adequately encapsulated in microspheres whose size is about 50–100 mm. Under sonication or homogenization, these parti- cles break down further into the 0.1–5 mm range suitable for encapsulation. Interestingly, the particles that break down smaller are smaller to start and are characterized by a finer, more friable microstructure ( Fig. 4 ). We hypothesize that the more friable microstructure is created during the spray-freez- ing step as smaller particles freeze faster creating smaller ice crystals. Upon drying, the smaller ice crystals are sublimated leaving behind the finer microstructure. In fact these powders have been shown to have a higher specific surface area indica- tive of the finer structure (14).

One possible disadvantage of the finer structure is the potential to affect the stability of the protein. Proteins can denature at the hydrophobic ice interface. We have observed

578 Tracy

Figure 4 The morphology of SFD protein powders prepared using different SFD processing parameters. The microstructure of the smaller-sized powder on the left is finer than that on the right. As

a result, under sonication, it breaks down into a proportionally smal- ler particle as indicated by the unsonicated =sonicated particle size ratios given. (The scanning electron micrographs are from Ref. 14.)

Figure 5 Improving integrity of SFD protein powders. The effect of zinc on the percentage of monomer loss is shown vs. median pro- tein particle size. (Adapted from Ref. 14.)

Biodegradable Microspheres for the Sustained Release of Proteins 579

a correlation between monomer loss, particle size, and specific surface area for BSA in the absence of any stabilizers. However, using the principle of minimizing mobility, proteins like BSA can be stabilized to prevent denaturation. For example, com- plexing BSA with zinc resulted in a significant enhancement in stability for submicron particles (14). Figure 5 shows that by adding zinc to BSA the monomer loss was greatly reduced in preparing protein particles for encapsulation by SFD. The effect was especially marked for the submicron protein parti- cles. Formulating with sugars also stabilized the protein in the small particles (15).

3.1. Summary The protein particle size and morphology impacts the release

from microspheres and protein stability. Process and formula- tion variables must both be balanced to optimize particle size, morphology, and stability to produce a microsphere product with optimal release characteristics.

4. CONCLUSIONS Two key themes have emerged from the development of biode-

gradable microsphere products for proteins. These themes apply in general to dry powder pharmaceutical products.

1. Maximize powder and drug stability by mini- mizing molecular mobility through formulation and processing.

2. Understand the effects of particle size and morphol- ogy on product function. Identify key process and formulation variables that affect product character- istics and control them.

REFERENCES 1. Tracy MA. Devolpment and scale-up of a microsphere protein

New York system. Biotechnol Progr 1998; 14:108–115.

580 Tracy 2. Creighton TE. Protein Structures and Molecular Properties.

New York: Freeman and Co., 1984. 3. Johnson OL, Cleland JL, Lee HJ, Charnis M, Duenas E,

Jaworowicz W, Shepard D, Shahzamani A, Jones AJS, Putney SD. A month-long effect from a single injection of microencap- sulated Human Growth Hormone. Nat Med 1996; 2: 795–799.

4. Johnson OL, Jaworowicz W, Cleland JL, Bailey L, Charnis M, Duenas E, Wu C, Shepard D, Magil S, Last T, Jones AJS, Putney SD. The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm Res 1997; 14:730–735.

5. Griebenow K, Klibanov AM. On Protein Denaturation in Aqueous-Organic Mixtures but not in Pure Organic Solvents. J Am Chem Soc 1996; 118:11695–11700.

6. Putney SD, Burke PA. Improving protein therapeutics with sustained release formulations. Nat Biotechnol 1998; 16:1–6.

7. Cunningham BC, Mulkerrin MG, Wells JA. Dimeration of human growth hormone by Zinc. Science 1991; 253:545–548.

8. Hancock BC, Zografi G. Characteristics and significance of the amorphous state in pharmaceutical systems. J Pharm Sci 1997; 86:1–12.

9. Hancock BC, Shamblin SL, Zografi G. Molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures. Pharm Res 1995; 12:799–806.

10. Edwards DA, Hanes J, Caponetti G, Hrkach J, Ben-Jebria A, Eskew ML, Mintzes J, Deaver D, Lotan N, Langer R. Large porous particles for pulmonary drug delivery. Science 1997; 276:1868–1871.

11. Siegel RA, Langer R. Controlled release of polypeptides and other macromolecules. Pharm Res 1984; 1:2–10.

12. Bawa R, Siegel RA, Marasca B, Karel M, Langer R. An expla- nation for the controlled release of maromolecules from polymors. J Contr Rel 1985; 1:259–267.

Biodegradable Microspheres for the Sustained Release of Proteins 581 13. Saltzman WM, Langer R. Transport rates of proteins in porous

materials with known microgeometry. Biophys J 1989; 55:163–171.

14. Costantino HR, Firouzabadian L, Hogeland K, Wu C, Beganski C, Carrasquillo KG, Cordova M, Griebenow K, Zale SE, Tracy MA. Protein spray-freeze drying. Effect of ato- mization conditions on particle size and stability. Pharm Res 2000; 17:1374–1383.

15. Costantino HR, Firouzabadian L, Wu C, Carrasquillo KG, Griebenow K, Zale SE, Tracy MA. Protein spray-freeze drying.

2. Effect of formulation variables on particle size and stability. J pharm Sci 2002; 91:388–395.

SECTION IV: QUALITY ASSURANCE AND REGULATION

Injectable Dispersed Systems: Quality and Regulatory Considerations

JAMES P. SIMPSON MICHAEL J. AKERS Regulatory and Government Affairs,

Pharmaceutical Research and Zimmer, Inc., Warsaw, Indiana, U.S.A.

Development, Baxter Pharmaceutical Solutions LLC, Bloomington, Indiana, U.S.A.

1. INTRODUCTION AND SCOPE The aim of this chapter is to provide the reader with an

appreciation of the principles and requirements for register- ing and marketing injectable drug products as dispersed systems. These dosage forms offer unique characteristics having certain distinct advantages over more conventional solid and liquid sterile products. Such unique characteristics also present special challenges in the manufacturing and control of these dosage forms.

584 Simpson and Akers