Pilot Production Of A Staphalokinase Variant (RGD-Sak, DGR) And Preparation Of Its Sustained-release Microsphere

The staphylokinase variant (RGD-Sak, DGR) was rationally designed and constructed on the basis of protein engineering of staphylokinase. The variant DGR remarkably inhibited platelet aggregation and suffered no loss of fibrinolytic activity. Morever, the immunogenicity of the variant was greatly reduced in guinea pigs compared with that of wt-Sak. It was implied that DGR had higher thrombolytic efficacy compared with that of r-Sak and showed relative antithrombotic effect and antiplatelet aggregation activity in vivo. With these encouraging findings, high level expression and purification of DGR were studied at pilot scale. The low immunogenicity of DGR was conducive to its preventive effective in thromboembolic diseases. Staphyloinase and its variants had relatively short plasma half-lives and had to administrate by intravenous infusion. It is very hard to develop DGR as a preventive drug. The development of biodegradable polymeric microspheres is becoming a promising way to overcome this shortcoming. Thus, we developed the DGR-loaded microspheres. There was no report concerning microspheres containing staphylokinase and its variants up to now.DGR was successfully expressed as a soluble cytoplasmic protein in Escherichia coli JF1125. The expression level reached 50% of total bacteria protein in 30L fermentor. Subsequently the expressed DGR was purified employing a simple two-step chromatographic purification process developed at pilot scale. The final yield of purified protein was 220mg/L of bacterial culture. SDS-PAGE, IEF, and HPLC/MS analysis indicated that the purified DGR was almost completely homogenous. The specific activity of DGR was about 1.2×10⁵ AU/mg. The procedure established here was simple, stable, high effective and easy to be scaled up for industrial production.DGR-loaded microspheres were fabricated using a double emulsion-solvent extraction /evaporation technique. Prior to encapsulation, DGR was exposed to a water/dichloromethane emulsion, and its stability was tested in the presence or absence of various excipients in the water phase. Moderate ultrasonic treatment of DGR/dichloromethane mixtures caused approx. 84% DGR aggregation. Proteinrecovery could not be improved by the addition of tween 80, poly(ethylene glycol) (PEG) 400, sucrose, mannitol, but poly vinyl alcohol (PVA) significantly improved DGR recovery to >90%. The protective effect of PVA on the stability of DGR showed concentration-response relationship. The effects of PVA in the internal aqueous phase and NaCl in the external aqueous phase on the characteristics of the microspheres were investigated. NaCl in the external aqueous phase played a critical role in determining encapsulation efficiency, microsphere morphology and particle size distribution. The stability of DGR within microspheres from various preparations was tested and more than 90% DGR within microspheres was in active form when PVA was used as excipient. In vitro release test showed that DGR was released from PLGA microspheres in a sustained manner over 15 days. A large proportion of released DGR was inactive in the absence of PVA. On the contrary, when 2% PVA was added into internal aqueous phase, the released DGR was almost completely intact within 9 days. In vivo experiments showed that DGR released from microspheres sustained 5 days. We concluded that PLGA microspheres capable be an effective carrier for DGR and is promising as a new depot system.DGR release from the microspheres was incomplete since DGR within microspheres was denatured during release. How to inhibit the denaturation of DGR during release was explored. The acidic microclimate was an important factor for the denaturation of DGR. And Mg(OH)₂ nanoparticles added into the internal aqueous phase successfully inhibited the denaturation of DGR. Another factor leading to the loss of DGR activity within microspheres was non-specific adsorption of DGR on the surface of polymer by hydrophobic and ionic interaction.The secondary structure of DGR encapsulated in PLGA microspheres was quantitatively examined by Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement technique, Fourier deconvolution, was combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands and assigned to α-helix, β-sheet, Turn and irregular (random) structures. The changes of bands at 1651 cm^-1 and 1623cm^-1 after encapsulation were discussed.