Anion binding mediated precipitation of a peptibody.
OBJECTIVE
Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions.


METHODS
The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7-5.0), salt concentration (0-400 mM), and ion identity using a series of monovalent, Hofmeister anions (F(-), Cl(-), Br(-), I(-), ClO(4) (-), SCN(-)) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation.


RESULTS
PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN(-) approximately ClO(4) (-)>I(-)>Cl(-)>Br(-)>F(-)) was observed with "salting-in" anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4 degrees C but decreased upon storage at 25 degrees C and 37 degrees C; the loss in reversibility correlated with an increase in intermolecular beta-sheet content of the precipitate.


CONCLUSIONS
Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.
Saluja
Atul
A
Process & Product Development, 1201 Amgen Court West, MS AW2-D/3152, Seattle, Washington 98119-3105, USA.
Crampton
Shon
S
Kras
Eva
E
Fesinmeyer
R Matthew
RM
Remmele
Richard L
RL
Narhi
Linda O
LO
Brems
David N
DN
Gokarn
Yatin R
YR
D000838:Anions / Q000737:chemistry*; D011232:Chemical Precipitation; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared
D000838:Anions; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; C539318:peptibody A; D012965:Sodium Chloride