BioPro HIC BF shows the stronger retention of proteins due to the higher hydrophobicity of its stationary phase compared to the other commercially available HIC columns. This indicates that BioPro HIC BF could be preferred for the separation of low hydrophobic proteins.

Column	100 X 4.6 mmI.D.
Eluent	A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 0%B (0-1 min), 0-100%B (1-11 min), 100%B (11-15 min)
Flow rate	0.5 mL/min
Temperature	25ºC
Detection	UV at 280 nm
Injection	15 µL
Sample	1. Adalimumab (0.5 mg/mL) 2. Trastuzumab (0.5 mg/mL)

Analysis of oxidized MAb samples, an example for biopharmaceutical characterization, is shown here.
BioPro HIC BF having strong hydrophobic retention could be preferred for such analysis because the oxidized MAb variants elute earlier than the nonoxidized MAb.

Analysis of oxidized MAb

NISTmAb was treated with tert-butyl hydroperoxide (t-BHP) as an oxidant in order to promote the oxidation. The oxidized MAb was analyzed by using BioPro HIC BF column.
Under the ammonium sulfate condition (a), four peaks appeared at earlier elution times compared to the peak of the nonoxidized MAb, presumably due to the conformational changes via the oxidization of the methionine residues.
Under the sodium chloride condition (b), eight peaks appeared at earlier elution times　compared to the peak of the nonoxidized MAb. Such a better resolution was achieved with the shorter analysis time compared with under the ammonium sulfate condition (a).

Column	BioPro HIC BF 4 µm, 100 X 4.6 mmI.D.
Eluent	A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing salt B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 40-80%B (0-40 min), 80%B (40-45 min)
Flow rate	0.3 mL/min
Temperature	25°C
Detection	UV at 280 nm
Injection	5 µL (1.0 mg/mL)

Analysis of papain-digested oxidized MAb

The papain-digests of NISTmAb samples with and without the oxidization were analyzed by using BioPro HIC BF column.
The Fab and Fc fragments were characterized from the chromatogram of the papain-digested MAb.
In the chromatogram of the papain-digested oxidized MAb, the multiple peaks appeared at earlier elution times compared to the peaks assigned to the Fab and Fc fragments, and would be corresponding to the oxidized fragments.
According to the previous report*, the oxidized fragments elute earlier than the nonoxidized ones.
*Journal of Chromatography A, 2008, 1214, 81-89

Column	BioPro HIC BF 4 µm, 100 X 4.6 mmI.D.
Eluent	A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 40-80%B (0-10 min)
Flow rate	1.0 mL/min
Temperature	25°C
Detection	UV at 280 nm
Injection	5 µL (0.5 mg/mL)

BioPro HIC BF shows excellent peak shape even under high loading conditions. This leads to effective for laboratory-scale purification and detection of minor constituents by the large volume injection.

Column	BioPro HIC BF 4 µm, 100 X 4.6 mmI.D.
Eluent	A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 60%B (0-0.5 min), 60-100%B (0.5-7.5 min), 100%B (7.5-10 min)
Flow rate	1.2 mL/min
Temperature	30°C
Detection	UV at 280 nm
Sample	Humanized monoclonal IgG (2.5 mg/mL)