Evaluation of a novel agarose-based synthetic ligand adsorbent for the recovery of antibodies from ovine serum.
This paper evaluates a prototype agarose-based affinity adsorbent utilizing a bound synthetic ligand designed to replace Protein A as an IgG-affinity capture resin and compares its purification characteristics with four commercially available matrices for the recovery of polyclonal antibodies from crude hyperimmune ovine serum. The novel adsorbent was found to show the highest dynamic capacity (29.2 mg/mL) of all matrices under evaluation--30% higher than the other commercial adsorbents evaluated. When using a post-load caprylic acid wash, IgG yields of over 85% and purities of over 90% were achieved consistently over multiple loading cycles. To evaluate bead diffusion, inverted confocal microscopy was used to visualise fluorescent antibody binding on to individual adsorbent beads in real time. The results indicate that the binding characteristics of the prototype adsorbent are similar to those obtained with Protein G Sepharose. This study indicates that the high-capacity prototype matrix is a feasible and potentially cost-effective alternative for the direct capture of antibodies from crude ovine serum and may therefore also be applicable to the purification of other complex industrial feedstocks such as transgenic milk or monoclonal antibodies expressed using recombinant technologies.
Chhatre
Sunil
S
The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom. sunil.chhatre@ucl.ac.uk
Francis
Richard
R
Titchener-Hooker
Nigel J
NJ
Newcombe
Anthony R
AR
Keshavarz-Moore
Eli
E
D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000097:blood / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008024:Ligands; D018613:Microscopy, Confocal; D012685:Sepharose / Q000737:chemistry*; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D000906:Antibodies; D008024:Ligands; D012685:Sepharose