Use of biophysical characterization in preformulation development of a heavy-chain fragment of botulinum serotype B: evaluation of suitable purification process conditions.
OBJECTIVE
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.
Bedu-Addo
Frank K
FK
Diosynth RTP, Inc, Cary, North Carolina 27513, USA.
Johnson
Catharine
C
Jeyarajah
Shanthini
S
Henderson
Ian
I
Advant
Siddharth J
SJ
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000302:isolation & purification*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D004364:Pharmaceutical Preparations; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature
D010446:Peptide Fragments; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D001905:Botulinum Toxins