Stability Of Protein Drugs Research Articles

Protein drug stability: a formulation challenge

The increasing use of recombinantly expressed therapeutic proteins in the pharmaceutical industry has highlighted issues such as their stability during long-term storage and means of efficacious delivery that avoid adverse immunogenic side effects. Controlled chemical modifications, such as substitutions, acylation and PEGylation, have fulfilled some but not all of their promises, while hydrogels and lipid-based formulations could well be developed into generic delivery systems. Strategies to curb the aggregation and misfolding of proteins during storage are likely to benefit from the recent surge of interest in protein fibrillation. This might in turn lead to generally accepted guidelines and tests to avoid unforeseen adverse effects in drug delivery.

A novel approach to stabilization of protein drugs in poly(lactic-co-glycolic acid) microspheres using agarose hydrogel

A novel approach has been taken to stabilize protein drugs in poly(lactic-co-glycolic acid) (PLGA) microspheres. This approach creates a new protein drug delivery system, which is based on the combination of agarose hydrogel particles and PLGA microspheres. This combination produces a heterogeneously structured polymeric composite. The protein drug molecules are encapsulated in the agarose hydrogel particles and the drug-containing agarose hydrogel particles are further dispersed in the PLGA microspheres. One PLGA microsphere may contain many agarose hydrogel particles to form a PLGA–agarose composite microsphere. The PLGA–agarose composite microspheres have spherical shape and a smooth surface. They possess a normal or Gaussian size distribution and an average diameter of 150 μm. The PLGA–agarose composite microspheres have higher protein loading efficiency than that of the conventional PLGA microspheres. The hydration of the PLGA–agarose composite microsphere matrix is faster than that of the conventional PLGA microspheres. Protein drugs can be slowly released from the PLGA–agarose composite microspheres. The agarose hydrogel particles can stabilize protein drugs in the PLGA matrix, which is the major advantage of this novel protein drug delivery system over the conventional PLGA microspheres.

Rapid enrichment of hematopoietic stem cells from human vertebral body marrow

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4 h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

The use of panel of monoclonal antibodies against different components of crude collagenase to study the function of collagenase in the digestion of the human pancreas

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4 h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

Is stability prediction possible for protein drugs? Denaturation kinetics of beta-galactosidase in solution.

Denaturation and aggregation kinetics of Aspergillus oryzae beta-galactosidase in solution were studied in order to determine whether the stability of protein drugs can be predicted. Denaturation of beta-galactosidase, monitored by measuring enzyme activity, conformed to first-order kinetics, whereas aggregation of the denatured form, monitored by high performance size exclusion chromatography, showed a reaction order higher than 1. Denaturation of beta-galactosidase was irreversible and exhibited a biphasic kinetic pattern which could be explained by assuming that two isoenzymes denatured irreversibly at different rates. Linear Arrhenius plots were obtained for the estimated rate constants, and delta H not equal to and delta S++ to were estimated according to the Eyring equation. The estimated delta H++ was much larger than delta H++ observed in usual chemical reactions. The present study suggests that the denaturation of protein drugs can be analyzed by the Eyring equation in the same manner as chemical degradation, contradicting the general consensus that accelerated testing can not be used to predict the stability of protein formulations.