Technical Information

Analysis of oxidized monoclonal antibodies by hydrophobic interaction chromatography

During manufacture and/or storage of biopharmaceuticals, variants with different properties from desired substances are produced by enzyme reactions or physicochemical interactions. Characterization of the variants is of great importance from the perspective of ensuring efficacy and safety of pharmaceutical products.
Oxidized mAb variants can be analyzed by hydrophobic interaction chromatography (HIC). On this page, we introduce the separation of mAb samples and their oxidized species using our HIC column, BioPro HIC BF.

mAb oxidation with t-BHP treatment

tert-Butyl hydroperoxide (t-BHP) was used as a chemical oxidant to promote oxidation of methionine residues of NIST mAb.
Subsequently, papain was used for the preparation of two Fab fragments and one Fc fragment.

Analysis of oxidized mAb

BioPro HIC BF column demonstrated the separation of oxidized mAb from the nonoxidized mAb using a low flow rate and shallow gradient slope.
In the chromatogram (a), earlier eluting four peaks were assumed to be derived from species that would have oxidized methionine residues on the mAb. The oxidation of sulfide side chains on methionine residues might result in conformational changes.
By using sodium chloride instead of ammonium sulfate, better resolution was achieved with a short analysis time (b).

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

Papain digested NIST mAb samples were analyzed. Fab and Fc peaks were observed on analysis of the nonoxidized mAb sample.
Peaks that would correspond to oxidized Fab and Fc were observed on the analysis of papain digested oxidized mAb sample. The oxidized species eluted earlier than the nonoxidized species.

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)

*Journal of Chromatography A, 2008, 1214, 81-89