Introduction
Antibodies (Abs) are routinely used as detection and quantitation agents
in scientific research and as vehicles for delivering radioisotopes,
drugs and enzymes to cells of interest in
medicine.1-2 It is the
inherent high specificity combined with the strong binding affinity
towards diverse antigens (proteins, carbohydrates, nucleotides and
haptens) that explains why personalized medicine relies to a great
extent on monoclonal antibodies (mAbs),3 many of which find
their use in cancer therapy.4-5 The striking success
of mAbs as efficient therapeutic agents resulted in 61 FDA- approved
mAbs by 2017 6 and
~ 570 mAbs are currently in advanced clinical trials.7 The increasing global
demand for diverse mAbs pressures pharmaceutical companies to improve
the expression levels of mAbs (upstream processing) and their
purification (downstream processing).
Antibody purification is most commonly achieved viachromatography using Protein A (ProA) as the ligand of choice for
capturing Abs. ProA binds strongly8 and specifically9 to diverse Ab
subclasses, 10 making
it possible to efficiently capture Abs from very complex media and to
reach high purity (>98%) within a single chromatographic
step. 11 These features
have thus made ProA chromatography the gold standard technology in Ab
purification. 12However, the necessity to purify ton amounts13 of therapeutic mAbs
has led pharmaceutical companies to search for a more economical route
to meet the above challenge. The ideal purification technology would
thus rely on a non-chromatographic step capable of efficiently capturing
Abs. This challenging objective was termed the: ”ABC philosophy”,i.e., Anything But Chromatography.12,
14 We sought to take this philosophy an
additional step forward by entirely excluding ProA from the process.
Development of a non-chromatographic, ligand-free Ab purification
platform has the potential of overcoming major problems related to ProA
chromatography. These include: (i) the high cost of ProA resins and the
potential leaching of ProA (or its fragments) into the purified Ab;12 (ii) the limited
binding capacities of ProA columns which may pose a problem at high Ab
concentrations; 15(iii) deamidation of ProA asparagines during column sanitation,16 leading to lower
binding efficiency; and (iv) the high maintenance costs associated with
the use of HPLC\FPLC instrumentation.
Great efforts have been directed toward replacing ProA chromatography
with a non-chromatographic approach. These included: (a) diverse
precipitation methods;17 (b) extraction
systems; 18-19 and (c)
ultrafiltration with charged membranes;20 but none of these
are widely used.
Recently, we described an alternative Ab purification platform that
complies with such challenging criteria.21-22 Our approach
relied on literature reports describing purification of mAbs viahydrophobic interaction chromatography (HIC)23-25 or hydrophobic
membrane interaction chromatography.26 Those studies
demonstrated the tendency of immunoglobulins (IgGs) to bind more
strongly to non-polar surfaces than do non-IgG water-soluble proteins.
We exploited this greater binding affinity of IgGs to hydrophobic resins
by demonstrating that Tween-20 detergent micellar aggregates can be used
for purification of human, mouse, rabbit and sheep IgGs.21-22
Tween-20 aggregates were formed in two-steps: first, the amphiphilic
chelator, bathophenanthroline (batho), was added to a dispersion of
Tween-20 micelles and transformed the latter into what we have termed:
‘engineered-micelles’,27-29 in which the
chelator is presumably positioned at the micelle/water interface. This
was followed by specific conjugation of engineered-micelles with
Fe2+ ions (known to bind with high affinity up to
three batho molecules in parallel14,
30) leading to Tween-20 aggregates as a
distinct amphiphilic phase. The latter were shown to efficiently capture
IgG’s from hybridoma serum-free media, or from E. coli lysate,
and allow extraction of pure target IgG’s without concomitant extraction
of impurities or aggregate dissolution.21-22
In the present report, we demonstrate the generality of our
non-chromatographic, ligand-free approach, as well as providing evidence
which would further justify efforts at up-scaling. Achieving these
challenging objectives is expected to form the basis for considering
integration of our purification platform into standard downstream
processing of therapeutic grade mAbs on an industrial scale.