Janet Yu-Feng Yang

A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.

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.

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.