Douglas A. Yeung

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

AbstractPurpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.

Surface Denaturation at Solid-Void Interface—A Possible Pathway by Which Opalescent Participates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures

During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 °C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22 - 1.78 m2/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103 - 105 °C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.

Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect

The treatment of growth hormone deficiency requires the daily administration of recombinant human growth hormone (rhGH). Long lasting formulations of rhGH have the potential to increase patient compliance, improve quality of life, and increase the efficacy of rhGH (lower total dose). One approach to these formulations is the use of biodegradable, injectable microspheres consisting of poly(lactic-co-glycolic acid) (PLGA). rhGH PLGA microspheres (12% w/w rhGH) were produced using a conventional double emulsion process. Initial in vitro studies of these microspheres indicated an initial release of 35% (1 day) and a continuous release for 21 days. A single administration of these microspheres (200 mg) in rhesus monkeys resulted in an initial elevation of serum hGH levels followed by a lag phase (return to baseline) until day 12 and then a sustained level of hGH greater than 5 ng/ml for 30 days. Development of improved in vitro release methods yielded release profiles comparable to those observed in vivo as determined from a detailed pharmacokinetic analysis of the hGH serum data. Serum hGH concentrations at or above 5 ng/ml provided a maximal response in two primary indicators of hGH biological activity, insulin-like growth factor-I (IGF-I) and IGF-binding protein 3. Further assessment of serum samples in an in vitro cell-based assay indicated that the rhGH released in vivo from the microspheres was bioactive. The rhGH PLGA formulations were well tolerated with mild to moderate inflammation and fibrosis. One of four animals developed a low titer anti-hGH antibody response, but transgenic mice expressing hGH did not develop an immune response to rhGH released from the PLGA microspheres. Therefore, this formulation had a low immunogenicity similar to the commercial rhGH formulation (Nutropin®). Overall, these studies demonstrated that a single administration of rhGH PLGA microspheres provide a prolonged release of bioactive rhGH that is well tolerated. With the improved in vitro release methods, future rhGH PLGA formulations may be designed without a lag phase to yield a one month continuous release of bioactive rhGH.