Preformulation development of recombinant pegylated staphylokinase SY161 using statistical design.

The goal of this study was to perform preformulation development of SY161 by using statistical design methods to understand the effects of buffer strength, NaCl concentration, and pH on conformation and stability of the protein. It was also important to elucidate interactions between these factors. A central composite design using a 2-level full-factorial study was performed. Secondary structure was evaluated using circular dichroism. Stability toward unfolding was investigated using high-sensitivity differential scanning calorimetry. Depegylation, aggregation, and protein loss were evaluated using SEC-HPLC with on-line light scattering, at time zero and after a 2-week stability study. Response surface plots clearly show optimal pH, NaCl, and buffer conditions. Interactions between pH and NaCl as well as pH and buffer concentration are observed. Tm is seen to be predictive of SY161 stability. Secondary structure changes were minimal and did not influence stability. Statistical design was very effective in providing an understanding of the effects of the formulation components on SY161 stability.

Formulation of botulinum neurotoxin heavy chain fragments for vaccine development: mechanisms of adsorption to an aluminum-containing adjuvant.

Heavy chain fragments of botulinum neurotoxin serotypes A and B are being developed as a bivalent vaccine for botulism. To potentiate the immune response, an aluminum containing adjuvant will be formulated with the two antigens. The adsorption mechanisms of each antigen to aluminum phosphate and aluminum hydroxide adjuvants were studied. The adsorption of the serotype A antigen to each adjuvant, and the serotype B antigen to aluminum phosphate adjuvant, is dependent on electrostatic attractive forces. The serotype A antigen is basic, and pretreatment with phosphate anions is required for favorable adsorption conditions to aluminum hydroxide adjuvant. In contrast, the serotype B antigen displays a high affinity to aluminum hydroxide adjuvant even when the two species possess the same charge. It is proposed that the serotype B antigen is adsorbed to aluminum hydroxide adjuvant by a ligand exchange mechanism.

Use of biophysical characterization in preformulation development of a heavy-chain fragment of botulinum serotype B: evaluation of suitable purification process conditions.

PURPOSE: The purpose of this study was to investigate the physicochemical and structural characteristics of recombinant botulinum serotype B (rBoNTB(Hc)) under various conditions and to use the information in evaluating suitable purification process conditions. METHODS: The solubility of rBoNTB(Hc) was evaluated at pH 4, 5, 6 7.5, 8, and 9. Secondary structure was evaluated using circular dichroism, and conformational stability was monitored using highsensitivity differential scanning calorimetry. Hydrophobic interaction chromatography, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), peptide mapping, and UV spectroscopy were used to monitor stability under the various conditions. RESULTS: The secondary structure of rBoNTB(Hc) consists predominantly of beta-sheets. Solubility of rBoNTB(Hc) was lowest at its pI and highest at low and high pH. In the presence of NaCl, however, solubility decreased with increase in pH. Conformational and chemical stability are improved below pH 7.5. In the presence of 150 mM NaCl at high pH, conformational and chemical stability of rBoNTB(Hc) are further decreased. The study suggests that the purification process should minimize exposure of rBoNTB(Hc) to high pH and salt conditions. CONCLUSIONS: Optimal stability of rBoNTB(Hc) is achieved at low pH. The biophysical and analytical studies provide us with an understanding of rBoNTB(Hc) stability behavior in solution and assists in developing efficient purification conditions.

Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.

Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.