Introduction
Monoclonal antibodies (mAbs) are powerful detection tools in science
[1, 2]
and valuable therapeutic agents in medicine
[3, 4].
Their significant contribution to human health derives from their strong
binding affinity and high specificity towards various antigens,
including proteins, carbohydrates, lipids and nucleotides, and , as
such, allow their use in personalized medicine
[5] and cancer therapy
[6, 7].
In 2021 alone, 16 new mAbs were expected to be approved by the American
Food and Drug Administration [8].
Clearly, as the global demand for diverse mAbs grows, increase in large
scale production and expression levels of mAbs (i.e. upstream
processing) combined with their purification (i.e. downstream
processing), is becoming ever more challenging
[9].
The majority of mAbs are purified from cell culture via Protein A
chromatography due to the high binding affinity
[10] and specificity
[11] of the Protein A ligand to a
wide range of antibody (Ab) isotypes
[12]. The unique properties of
Protein A translate into excellent recovery yields (generally
>90%) and high purity (>98%), both
accomplished within a single chromatographic step
[13], making Protein A chromatography
the gold standard technology in Ab purification
[14]. In general, mAbs are eluted
very efficiently from Protein A resins under acidic conditions
(e.g. 0.1 M sodium citrate, pH 3.3
[15]). Low pH is required for (a)
weakening the interactions between the immobilized Protein A and the Fc
domain of the target antibody; and for (b) inactivating any viruses
which may be present as impurities in the system
[16,
17].
However, low pH may also promote
disruption of antibody secondary structure which can lead to aggregate
formation [16-20].
Therefore, suppression or
elimination of antibody aggregation , during both upstream and
downstream processing, has become a subject of on-going investigation in
bioprocess development [20]. The high
antibody concentration present in the cell culture medium increases the
frequency of protein self-adsorption events
[16, 21,
22], which, of course, can also lead to
aggregation. Several strategies and/or small molecule additives, aimed
at minimizing antibody aggregation during column elution, have been
tested. They have included for example: (a) addition of 0.5-1M urea
[16] or (b) of arginine monomers
[21]
to the running buffer. Suppressing
the formation of antibody oligomers is vital: studies have shown that
such aggregates may reduce (i) therapeutic antibody potency; (ii) batch
to batch reproducibility; as well as (iii) promoting undesirable
immunogenic response [18,
23-25].
We have studied an alternative purification method that would avoid
exposing polyclonal human IgG’s to acidic conditions. This approach
relies on a recently described non-chromatographic, ligand-free strategy
that uses nonionic detergent micelles conjugated via the
amphiphilic complex
[(bathophenanthroline)3:Fe2+]
[26-28] as the purification platform.
Such conjugated detergent micelles were found to quantitatively capture
human and mouse IgG’s at neutral pH, exclude hydrophilic protein
background impurities and allow extraction of relatively pure IgG’s
without parallel co-extraction of background impurities or dissolution
of the conjugated micelles [26-28].
The approach was found to function efficiently with three commercially
available surfactant families characterized by polyethylene oxide (PEO)
headgroups: Tween; Brij and Triton X-100. We note that PEO-based
surfactants are approved for use in pharmaceutical formulations.
However, it was found to be necessary to lower the pH to 3.8 during IgG
extraction in order to achieve overall satisfactory yield of pure
antibodies (generally >80%,
[28]). Here, we investigated the
possibility of weakening the interactions between captured antibodies
and the detergent matrix to such an extent that antibody capture would
not be affected while efficient extraction could be performed at close
to neutral pH.