Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders using Supercritical or Near-Critical Fluids

Supercritical or near-critical fluid processes for generating microparticles have enjoyed considerable attention in the past decade or so, with good success for substances soluble in supercritical fluids or organic solvents. In this review, we survey their application to the production of protein particles. A recently developed process known as CO2-assisted nebulization with a Bubble Dryer® (CAN-BD) has been demonstrated to have broad applicability to small-molecule as well as macromolecule substances (including therapeutic proteins). The principles of CAN-BD are discussed as well as the stabilization, micronization and drying of a wide variety of materials. More detailed case studies are presented for three proteins, two of which are of therapeutic interest: anti-CD4 antibody (rheumatoid arthritis), α1-antitrypsin (cystic fibrosis and emphysema), and trypsinogen (a model enzyme). Dry powders were formed in which stability and activity are maintained and which are fine enough to be inhaled and reach the deep lung. Enhancement of apparent activity after CAN-BD processing was also observed in some formulation and processing conditions

Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state

The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing β-lactoglobulin (βLg):mannitol (1∶1–1∶15 wt/wt) were vacuum dried at 5°C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1∶5 wt/wt βLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1∶1 wt/wt βLg:mannitol dried sample. FITR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1∶5 wt/wt) βLg:mannitol ratios. The Tg of the dried 1∶1 wt/wt βLg:mannitol sample was observed at 33.4°C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the βLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1∶1 wt/wt in all dried samples. Second-derivative FTTR studies on dried βLg and recombinant human interferon α-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein

Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: Development of a novel process for obtaining a dry, stable powder

Feasibility studies were performed on the development of a novel process based on polyethylene glycol (PEG)-induced precipitation of proteins followed by vacuum drying in the presence of sugars to obtain dry protein powders. Apparent solubility of interferon alpha-2a (IFNα2a) was determined in the presence of various PEGs and the effect of solution pH, ionic strength, and temperature was investigated. IFNα2a precipitate was dried at a shelf temperature of 25°C at 100 mTorr either as it is or in the presence of mannitol and/or trehalose. The dried IFNα2a formulations were subjected to accelerated stability studies at 40°C (3 months), and the stability was compared with that of a similar lyophilized formulation. The results indicated that more than 90% of the protein could be precipitated using 10% wt/vol PEG the protein could be precipitated using 10% wt/vol PEG 1450 at pH 6.5 at a solution ionic strength of 71 mM. Vacuum drying of the precipitate only resulted in the formation of insoluble aggregates of IFNα2a; however, this was prevented by the addition of either mannitol or trehalose. The addition of excess mannitol resulted in low residual moisture content and better handling of the final dried product. Accelerated storage stability did not show any aggregation and showed less than 5% formation of oxidized IFNα2a in the dried formulation containing IFNα2a: trehalose: mannitol in a 1∶10∶100 wt/wt ratio upon storage at 40°C for 3 months. The stability of this vacuum dried formulation was comparable with that of a similar lyophilized formulation