Role of amphiphilic [metal:chelator] complexes in a non-chromatographic antibody purification platform

Dhandapani, Gunasekaran; Nair, Divya K.; Kale, Raju R.; Wachtel, Ellen; Namboothiri, Irishi N. N.; Patchornik, Guy

doi:10.1016/j.jchromb.2019.121830

Cited by 9 publications

(8 citation statements)

References 22 publications

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“…Three previous studies [27][28][29] demonstrated that efficient antibody purification is achieved only when non-ionic detergent micelles form a new and larger oil-rich phase upon micellar conjugation with the [(bathophenanthro line) 3 :Fe 2+ )] complex. This suggests that van der Waals and/or entropically driven hydrophobic interactions are at the heart of the IgG binding mode in our purification strategy.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently presented a novel purification platform for intact immunoglobulin G (IgG) that does not rely on chromatographic media or ligand [27][28][29] . This approach makes use of non-ionic detergent (e.g.…”

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confidence: 99%

“…2 Faculty of Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel. * email: guyp@ariel.ac.il detergent aggregates were shown to quantitatively capture diverse IgG's from human, mouse, rabbit, or sheep sera, reject hydrophilic proteins and allow extraction of captured IgG's from the aggregates without concomitant impurity co-extraction or aggregate dissolution [27][28][29] .…”

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confidence: 99%

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Purification of antibody fragments via interaction with detergent micellar aggregates

Dhandapani

Wachtel

Das

et al. 2021

Sci Rep

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The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab′)2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab′)2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab′)2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab′)2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab′)2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG’s as test samples.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Purification of antibody fragments via interaction with detergent micellar aggregates

Dhandapani

Wachtel

Das

et al. 2021

Sci Rep

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“…The objective of this communication is to present an alternative purification method that would be straightforward to implement, nondenaturing, and would not be compromised by the large size and small diffusion coefficient of IgMs. Accordingly, we studied a nonchromatographic, ligand‐free method that has demonstrated its utility with IgGs and F(ab′) 2 fragments: the active medium is based on micellar aggregates that are formed upon conjugation with amphiphilic [(bathophenanthroline) 3 :Fe 2+ ] complexes (Dhandapani, Howard, et al, 2019; Dhandapani, Nair, et al, 2019; Dhandapani et al, 2020, 2021) Such aggregates were found to: (i) quantitatively capture IgGs (Dhandapani, Howard, et al, 2019; Dhandapani, Nair, et al, 2019; Dhandapani et al, 2020), as well as the F(ab′) 2 domain of a monoclonal antibody (Dhandapani et al, 2021) (presumably due to hydrophobic interactions with the detergent aggregates, in agreement with diverse studies showing how IgGs are purified via hydrophobic interaction chromatography) (Follman & Fahrner, 2004; Ghosh & Wang, 2006; Guse et al, 1994; Manzke et al, 1997); (ii) reject hydrophilic impurities; and (iii) allow efficient recovery of pure antibodies from the detergent aggregates at pH 3.8.…”

Section: Introductionmentioning

confidence: 99%

Conjugated detergent micelles as a platform for IgM purification

Dhandapani

Wachtel

Das

et al. 2022

Biotech & Bioengineering

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Immunoglobulin M (IgM) antibodies hold promise as anticancer drugs and as agents for promoting immune homeostasis. This promise has not been realized due to low expression levels in mammalian cells producing IgM class antibodies, and the failure of protein A chromatography for IgM purification. Here, we describe a nonchromatographic platform for quantitatively capturing IgMs at neutral pH, which is then recovered with 86%-94% yield and >95% purity at pH 3. The platform contains micelles conjugated with the [(bathophenanthroline) 3 :Fe 2+ ] amphiphilic complex.Inclusion of amino acid monomers, for example, phenylalanine or tyrosine, during conjugation of detergent micelles, allows subsequent extraction of IgMs at close to neutral pH. With the successful implementation of this purification platform for both polyclonal humans and bovine IgMs, we anticipate similar results for monoclonal IgMs, most relevant for the pharmaceutical industry.

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“…We recently reported that aggregates with size ranging between 100-200 nm do not efficiently capture IgG. 22 It was therefore surprising to find that DLS characterized Pluronic F-127 aggregates as having the largest size (1312 nm) of all the detergents we studied: i.e., Tween (355 -685 nm), Brij (635 -683 nm) , Triton X-100 (913 nm) (Figure 3, A-D) . Thus, the larger size of Pluronic F-127 aggregates apparently could not compensate for the block copolymer core/shell micelle structure.…”

Section: Resultsmentioning

confidence: 84%

Nonionic detergent micelle aggregates: an economical alternative to Protein A chromatography

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We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed: [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates lead to high purity of the recovered Abs (>95%, by gel densitometry). Good overall Ab recovery yields were observed with: Tween-20 (80-83%), Brij-O20 (85-87%) and Triton X-100 (87-90%), while Pluronic F-127 was less efficient (42-53%). Of additional importance is the finding that the process can depend on filtration (rather than centrifugation), thereby allowing a continuous purification mode that leads to the recovery of monomeric IgG's (by DLS) and preservation of Ab specificity (by ELISA). The amphiphilic chelator, bathophenanthroline (batho) is recycled almost quantitatively (95%) by crystallization. Good IgG recovery yields (˜80%) are also observed when Ab concentrations are increased from 1 mg/ml to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies (mAbs), are discussed.

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Role of amphiphilic [metal:chelator] complexes in a non-chromatographic antibody purification platform

Cited by 9 publications

References 22 publications

Purification of antibody fragments via interaction with detergent micellar aggregates

Purification of antibody fragments via interaction with detergent micellar aggregates

Conjugated detergent micelles as a platform for IgM purification

Nonionic detergent micelle aggregates: an economical alternative to Protein A chromatography

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