Systematic DOE Approach for Dry Granulation Study at Early Clinical Stage of Novel Drugs: An Industrial Case.

In order to introduce a cost-effective strategy method for commercial scale dry granulation at the early clinical stage of drug product development, we developed dry granulation process using formulation without API, fitted and optimized the process parameters adopted Design of Experiment (DOE). Then, the process parameters were confirmed using one formulation containing active pharmaceutical ingredient (API). The results showed that the roller pressure had significant effect on particle ratio (retained up to #60 mesh screen), bulk density and tapped density. The roller gap had significant influence on particle ratio and specific energy. The particle ratio was significantly affected by the mill speed (second level). The tabletability of the powder decreased after dry granulation. The effect of magnesium stearate on the tabletability was significant. In the process validation study, the properties of the prepared granules met the requirements for each response studied in the DOE. The prepared tablets showed higher tensile strength, good content uniformity of filled capsules, and the dissolution profiles of which were consistent with that of clinical products. This drug product process development and research strategies could be used as a preliminary experiment for the dry granulation process in the early clinical stage.

Recycling reaction and separation for FACylation of loxenatide by trade-off between miscibility and immiscibility of reactants and product in methanol solution.

The growing demand and scale of production for fatty acid chain modified (FACylated) polypeptide has sparked the interest in novel production technologies. In this study, a recycling reaction and separation process was proposed and applied to the fatty acid chain modification (FACylation) of loxenatide (LOX), which was based on the difference in solubility between reactants and FACylated product. Especially, the mixed PBS-Methanol (MeOH) solution was designed to meet the demands for FACylation of LOX as well as separation of FACylated LOX and residual modifier. In order to ensure the efficient FACylation, a mixed 10% PBS-90% MeOH (v/v) solution was chosen to provide a good miscibility for two reactants, LOX and N-tetradecylmaleimide (C14-MAL). On the other hand, the immiscibility between reactant (C14-MAL) and FACylated product (N-tetradecyl-Loxenatide (C14-LOX)) could realize the separation of C14-LOX when the MeOH concentration was less than 30% (v/v). Based on this strategy, the recycling reaction and separation process for FACylation of LOX was established by adjusting the MeOH concentration in the mixed solution. The reaction yield and recovery of C14-LOX exceeded 97% and 94%, and the excess reactant C14-MAL could be recycled with a recovery of more than 80%. Furthermore, after purification by reversed-phase chromatography, C14-LOX showed good pharmacokinetic and pharmacodynamic properties in vivo. This study will have great application prospects in industrial production of C14-LOX.

Combined optimization of N-terminal site-specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis.

In this study, a combined optimization method was developed to optimize the N-terminal site-specific PEGylation of recombinant hirudin variant-2 (HV2) with different molecular weight mPEG-propionaldehyde (mPEG-ALD), which is a multifactor-influencing process. The HV2-PEGylation with 5 kDa mPEG-ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono-PEGylated product using combined statistical methods, including the Plackett-Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG-ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono-PEGylated-HV2 was modified at the N-terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N-terminal site-specific PEGylation process to obtain a homogeneous mono-PEGylated protein with desirable yield.