CHARACTERIZATION OF MAB AFFINITY ON FCγRIIIALIGAND BY ANALYTICAL AND SEMI-PREPARATIVE CHROMATOGRAPHY

Binding between monoclonal antibodies (mAbs) and effector cells directly impacts the accompanying immune response or antibody-dependent cell cytotoxicity (ADCC). N-glycans at Asn297 of the Fc region influence this binding, as they affect the specificity of IgG to the FcγRIIIA receptor of effector cells. To monitor Fc receptor binding, a recombinant FcγRIIIa-ligand has been commercialized into TSKgel FcR-IIIA high performance affinity chromatography (AFC) columns that separate antibodies based on affinity differences to FcγRIIIa.

TSKgel FcR-IIIA-NPR has a linear loading range of 5-50 μg mAb, which is useful for analytical applications but is highly limiting if collection of elution products is necessary for further purification or analyses. This is overcome by the semi-preparative TSKgel FcR-IIIA-5PW column, allowing collection of sufficient material for improved analytical workflows in a short time.

Recombinant FcγRIIIa-ligand, with enhanced physiochemical stability, is immobilized onto a non-porous particle for analytical applications and onto a larger porous particle for semi-preparative applications. While both columns share the separation mechanism, theTSKgel FcR-IIIA-5PW achieves an increased loading capacity and elution volume.

Here, intact mAb and papain-digested mAb were used to compare the scalability of semi-preparative TSKgel FcR-IIIA-5PW and to provide a methodology to broaden from analytical to semi-preparative mode while the impact on resolution between the two different capacity columns is limited.

MATERIAL AND METHODS TSKgel FcR-IIIA Conditions

Columns: TSKgel FcR-IIIA-5PW, 10 μm, 7.8 mm ID × 7.5 cm TSKgel FcR-IIIA-NPR, 5 μm, 4.6 mm ID × 7.5 cm

Mobile phase: A: 50 mmol/L citrate/NaOH, pH 6.0 B: 50 mmol/L citrate/NaOH, pH 4.0

Method: Equilibrate: 5 CV mobile phase A Wash: 4 CV 25% mobile phase B Elution for

TSKgel FcR-IIIA-5PW: linear gradient 25-90% B over 14 CV; hold 4 CV at 90% B and 100% B Elution for TSKgel FcR-IIIA-NPR: linear gradient 0-100% B over 15 min

Flow rate: TSKgel FcR-IIIA-5PW: Equilibration, load, and wash steps: 0.5 mL/min Elution and hold steps: 0.25 mL/min TSKgel FcR-IIIA-NPR: 0.5 mL/min

Instrument: ÄKTA™ avant 25 FPLC Detection: UV @ 280 nm

Temperature: ambient

Sample: protein A-purified trastuzumab (Herceptin® biosimilar)

Load: 2.7 mg (TSKgel FcR-IIIA-5PW) 54 μg (TSKgel FcR-IIIA-NPR)