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A general platform for antibody purification utilizing engineered-micelles.

mAbs (2019-01-09)
Gunasekaran Dhandapani, Assaf Howard, Thien Van Truong, Thekke V Baiju, Ellina Kesselman, Noga Friedman, Ellen Wachtel, Mordechai Sheves, Dganit Danino, Irishi N N Namboothiri, Guy Patchornik
ABSTRACT

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated "Engineered-micelles" built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe2+ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70-80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
L-Arginine, reagent grade, ≥98%
Sigma-Aldrich
L-Glutamic acid, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
L-Leucine, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Iron(II) sulfate heptahydrate, ReagentPlus®, ≥99%
Sigma-Aldrich
L-Valine, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
L-Aspartic acid, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Polysorbate 20, tested according to Ph. Eur.
Sigma-Aldrich
Albumin from human serum, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
L-Histidine, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Anti-Bovine Albumin antibody produced in rabbit, whole antiserum
SAFC
EX-CELL® 610-HSF Serum-Free Medium for Hybridoma Cells, Low-protein, with L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
L-Isoleucine, 99%, FCC, FG