Optimised affinity purification of polyclonal antibodies from hyper immunised ovine serum using a synthetic Protein A adsorbent, MAbsorbent® A2P

Purification of antibodies using the synthetic affinity ligand absorbent MAbsorbent A2P

“…A representative chromatogram with absorbance (280 nm), pH and conductivity traces is shown in Figure 1. IgG purities (as determined by Coomassie-stained SDS-PAGE and scanning densitometry) 10 will be 90% (Fig. 2).…”

“…The purification protocol was developed after identifying critical parameters and optimizing process conditions using factorial experimental design 10 . Although similar protocols describing the purification of polyclonal antibodies from serum using alternative synthetic affinity absorbents have recently been described 11 , a major technical challenge to overcome when isolating antibodies from serum is the removal of nonspecifically bound serum proteins (particularly serum albumin) from the absorbent before IgG elution.…”

“…Increased back pressure may be due to absorbent compression resulting from ionization of the bound ligand affecting the rigidity of the agarose base matrix. This may in turn impact the performance of the absorbent for the purification of antibodies from serum using large-diameter commercial-scale chromatography columns 12 Although the protocol described herein has previously been evaluated for the purification of antibodies for the production of antibody-derived biotherapeutics 10 , this approach may be equally applicable for the purification of monoclonal antibodies for diagnostic or analytical techniques and may therefore be adapted for use in a wider range of applications. The protocol may also be applied for the purification of normal (non-hyperimmune) plasma antibodies and use with other chromatography absorbents utilizing small molecule ligands, such as hydrophobic charge induction chromatography 14,15 .…”

Purification Systems Based on Bacterial Surface Proteins

“…PAM was selected from a combinatorial peptide library, and to further increase the stability of this molecule D-amino acids have been used to hinder degradation of the molecule by proteases (Verdoliva et al 2002). A synthetic protein called MAbsorbentA2P (ProMetic BioSciences), which binds all subclasses of human IgG, has also been described (Newcombe et al 2005). One can also use thiophilic ligands for antibody purification, the most common is called "T-gel", which carries linear ligands with two sulfur atoms and displays good selectivity for antibodies in the presence of high concentrations of lyotropic salts (Boschetti 2001).…”

Screening and chromatographic assessing of a novel IgG biomimetic ligand

“…PBS at pH 7.4 with stepwise ionic strength of 0.3, 0.5, 0.8 and 1.0 M NaCl. Then a second stepwise series of 0.1 M sodium citrate solutions with pH values of 4.7, 3.5, 3.3, 3.0, 2.7 and 2.3 were applied (Newcombe et al, 2005;Daniela et al, 2004). The separation and purification of the diluted plasma proteins were carried out on an ÄKTA purifier (Amersham).…”