3 years ago

An engineered Staphylococcal Protein A based ligand: Production, characterization and potential application for the capture of Immunoglobulin and Fc-fusion proteins

Martin Kangwa, Vikas Yelemane, Adilah Ponnurangam, Marcelo Fernández-lahore

Publication date: Available online 13 November 2018

Source: Protein Expression and Purification

Author(s): Martin Kangwa, Vikas Yelemane, Adilah Ponnurangam, Marcelo Fernández-Lahore

Abstract

In antibody purification processes, affinity chromatography has been used with Staphylococcus aureus protein A (SpA) as the main ligand. In this work, we present a novel Staphylococcal Protein A (AviPure thereafter), a synthetic ligand analogue based on native SpA B domain, with a molecular weight of approximately 14 kDa.

The binding affinity of mAbs to AviPure was evaluated using Surface Plasmon Resonance (SPR) and affinity chromatography methods. The equilibrium dissociation constant (KD) between the AviPure and mAbs was systematically measured using 1:1 (Langmuir) model and found to be 4.7 × 10−8 M, with constant of dissociation at kd ≤ 1.0 × 10−3 s−1 and ka being 3.1 × 104 M−1 s−1. When immobilized on Sepharose, the AviPure ligand density was 429 nmol/g moist weight resin and was able to effectively bind immunoglobulin and Fc fragment samples with higher affinity and the most effective flow rate when using ligand - Sepharose beads was at 75 cm/h giving the dynamic binding capacity of 53 mg/mL and 91% recovery of IgG.

Suitable ligands used in affinity purification should have a KD ≤ 10−6 M and a dissociation rate (ka) averaging 10−3 M−1 s−1 with the kd ranging between 103 – 108 M−1. Therefore, the AviPure ligand can be used as an alternative to the standard protein A ligand in the purification of mAbs and Fc-fused proteins.

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