GUIDELINE #1: MINIMIZE MOLECULAR MOBILITY TO MAXIMIZE STABILITY

2. GUIDELINE #1: MINIMIZE MOLECULAR MOBILITY TO MAXIMIZE STABILITY

An excellent example of this principle is provided by the formulation and processing of biodegradable microspheres containing proteins (1). Proteins are relatively fragile macro- molecules whose tertiary structure is important for activity. The free energy difference between the native and unfolded state at room temperature is only of the order of the strength of one hydrogen bond (a few kilocalories =mol) (2). Therefore, it

Biodegradable Microspheres for the Sustained Release of Proteins 573

does not take much energy input at room temperature to disrupt a protein’s native state. Thus, to produce pharmaceu- tical products for these drugs, formulation and processing approaches are required which minimize the molecular mobi- lity during processing, after administration, and during sto- rage.

A process has been developed and commercialized to make a long-acting form of the protein human growth hormone (hGH) in which the protein is encapsulated in biode- gradable polymeric microspheres (Fig. 1) (1,3,4). The process first involves preparing solid particles of the protein, for exam- ple, by lyophilization. The particles are encapsulated by sus- pending them in a polymer solution containing an organic solvent, spray-freezing the suspension into liquid nitrogen to form nascent microspheres, extracting the polymer solvent in a polymer non-solvent at sub-zero temperatures, and finally drying the product under vacuum to minimize residual solvents.

The solid state form of the protein can be created by spray freeze-drying (SFD). The resulting intermediate is a dry powder itself in which protein mobility is reduced by the removal of water. This powder is exposed to organic

Figure 1 ProLease Õ encapsulation process steps.

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solvents during the encapsulation process. Protein native structure is not thermodynamically favored in organic sol- vents. Protein stability is maintained in organic solvents because the solid state provides a kinetic trap that maintains the stability (5,6). Key process factors that secure the kinetic trap are the low processing temperature and an anhydrous environment.

The encapsulation process keeps protein mobility mini- mized by encapsulating the protein in the solid state at low temperatures in the absence of water. Furthermore, the low processing temperature, well below the glass transition tem- perature of the polymer, minimizes polymer relaxation as well after the microspheres are formed.

Maintaining drug stability and activity after administra- tion is another challenge since the drug has to remain intact for the duration of release (days to months) at physiological conditions (37

C, pH 7). Maintaining protein stability over prolonged time periods has been achieved again using the principle of minimizing mobility. One approach for some pro- teins, including hGH and others, is to complex them with metal ions such as zinc (3,4,6,7). Zinc-protein complexes are more stable to denaturation (7). Figure 2 shows size exclusion chromatograms for hGH released from microspheres. The integrity of the protein released from microspheres containing zinc-hGH was similar to unencapsulated hGH and better than hGH released from microspheres without zinc (3,4). In essence the zinc binds to the protein forming a solid precipi- tate which provides the protein in a more rigid, longer lasting form. Another approach is to use salting out additives like ammonium sulfate (6).

Pharmaceutical products are typically required to have a shelf life of 2 years at the target storage temperature. For most products this is either 2–8

C or room temperature. In order to maximize storage stability, it is important to mini- mize molecular mobility. This can be accomplished by storing the product sufficiently below its glass transition temperature

(T g ) to prevent interactions that can adversely affect the pro- duct stability over short time periods such as days or weeks to longer periods of months or years. The rule of thumb is to

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Figure 2 Effect of zinc complexation on hGH stability during release from microspheres in vitro. The top chromatogram repre- sents unencapsulated hGH, the middle hGH released from micro- spheres with the stabilizer zinc added to form a zinc–hGH complex, and the bottom hGH released without complexing with zinc. The largest peak represents hGH monomer. Peaks to the left of it represent hGH dimers and oligomers. The buffer contained

50 mM HEPES and 10 mM KCl, pH 7.2. Release was carried out at 37 C. (Adapted from Ref. 3.)

store the product at least 30–50

C below its T g (8,9). For microsphere products, the polymer, poly(lactide-co-glycolide), is the major component and has a T g about 40

C. In manufac- turing, residual solvents in sufficient amounts can act as plasticizers and reduce the T g promoting mobility. A reduction in T g can impact the product stability during storage. It is thus important to minimize residual organic solvents in micro- sphere products to minimize mobility and maximize the pro- duct shelf life. This can be accomplished by developing a suitable process for drying microspheres. Protein and peptide microsphere products have been developed with shelf lives of at least 2 years.

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2.1. Summary For protein microsphere products, stability is maximized by

formulating the protein in the solid state, encapsulating under

a low temperature, anhydrous environment, and minimizing residual solvents. All of these steps have the goal of minimiz- ing the mobility of the protein or polymer during preparation, administration, and storage.