Mechanism of antibodies purification by protein A

Zarrineh, Mahshid; Mashhadi, Ilnaz Soleimani; Farhadpour, Mohsen; Ghassempour, Alireza

doi:10.1016/j.ab.2020.113909

Cited by 41 publications

(17 citation statements)

References 76 publications

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“…A similar observation has been made with other IgG studies in our group . Nevertheless, even if we only focuse on the recovered antibody mass, the huge amount of hIgG still exceeds the state of the art of reported capacities onto functionalized nanoparticles, which range between 100 and 150 mg g –1 − as well as reported data for microparticles, with binding capacities in the range of 30–100 mg g –1 . ,− This result confirms the basic idea of immobilized ligands on BION as a high promising adsorbent for antibody purification.…”

Section: Resultssupporting

confidence: 87%

“…They are widely commercially available even though they are usually offered for small scales. Their IgG binding capacities are in the range of 30–100 mg g –1 or 10–260 μg mL –1 slurry, respectively. − However, also magnetic particles of nanometer sizes can be used for protein separation: First studies indicate that the application of nano-sized magnetic particles for HGMS promises high product yields . Their small size in the nanometer range (8–15 nm) results in a large specific surface area of ∼90 m 2 g –1.…”

Section: Introductionmentioning

confidence: 99%

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Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Kaveh-Baghbaderani

Allgayer

Schwaminger

et al. 2021

ACS Appl. Nano Mater.

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The demand for purified antibodies is ever-rising. This study presents a nanoparticle-based material for efficient magnetic separation of immunoglobulin G (IgG) with high binding capacity. The characteristics include: (i) Cost-effective bare iron oxide nanoparticles are used as the solid phase on which optimized protein A-based ligands are directly immobilized. An additional chemical modification or activation is not needed. (ii) Oriented immobilization of the ligands is promoted using a C-terminal peptide tag, containing amino acids with an affinity for iron oxide. (iii) The immobilized ligand consists of eight polymerized B-domains of Protein A. This affinity adsorbent for antibody capture allows a recovery of up to 418 mg IgG per gram of particle, which exceeds the state of the art of magnetic nanoparticles as well as microparticles. Particles with a lower ligand density show a higher percentage recovery of IgG, which allows for a cost-effective design of the adsorbent. Furthermore, the selectivity of the immobilized ligand is shown by means of purification of rabbit polyclonal IgG using rabbit serum.

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Section: Resultssupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Kaveh-Baghbaderani

Allgayer

Schwaminger

et al. 2021

ACS Appl. Nano Mater.

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“…A number of artificial and pseudo-biospecific ligands on chromatography matrices have been developed to confer specificity and selectivity toward antibodies, thereby leading to the binding of antibodies to the matrix surfaces. Examples of this type of chromatography are affinity chromatography using artificial protein A or protein A mimetic ligands, thiophilic chromatography, immobilized metal affinity chromatography (referred to as IMAC, MCIC, or MCAC), , hydrophobic charge-induction chromatography (HCIC), − and cation-exchange chromatography using phosphate-immobilized zirconia. − The artificial ligands on the chromatography matrices are generally stable and cost-efficient but are less specific and less selective for antibodies than protein A . Accordingly, realization of high-purity purification using these artificial ligands as promising alternatives is a critical step for more widespread use of antibodies .…”

Section: Introductionmentioning

confidence: 99%

“…15−18 The artificial ligands on the chromatography matrices are generally stable and cost-efficient but are less specific and less selective for antibodies than protein A. 19 Accordingly, realization of high-purity purification using these artificial ligands as promising alternatives is a critical step for more widespread use of antibodies. 20 A promising strategy for this is to control the binding modes and binding capacities of antibodies by designing a mobile phase on the basis of a binding mechanism that remains unclear.…”

Section: ■ Introductionmentioning

confidence: 99%

Interactions between Amino Acids and Zirconia Modified with Ethylenediaminetetra(methylenephosphonic acid): Mechanistic Insights into the Selective Binding of Antibodies

et al. 2021

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Zirconia modified with ethylenediaminetetra(methylenephosphonic acid) (EDTMP) has an affinity for antibodies, including immunoglobulin G (IgG) and immunoglobulin M (IgM). However, little is known about the mechanism underlying antibody selectivity. In this study, we examined the interactions of EDTMP-modified zirconia with proteinogenic amino acids using chromatographic and batch methods to gain mechanistic insights into antibody selectivity at the amino acid level. We demonstrated that EDTMP-modified zirconia has an affinity for amino acids with a positively charged side chain, especially lysine. Similar trends were observed for oligopeptides. This affinity was reduced by the addition of sodium phosphate or sodium polyphosphates. Thus, the antibody selectivity of EDTMP-modified zirconia is primarily ascribable to electrostatic attractions between the EDTMP moieties of the zirconia surfaces and the constant region of antibodies that are rich in lysine residues. Consistent with this, the human IgG antibody has a higher adsorption ability onto EDTMP-modified zirconia than the rabbit IgG antibody, which has fewer lysine residues in the constant region. These findings are useful not only for improving antibody purification but also for developing new applications, including purification of proteins tagged with positively charged amino acid residues.

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“…It can also bind to immune complexes and immunoglobulins in blood and serum, which has been widely used in biotechnology. Immobilized SpA on polymeric supports can be used as affinity chromatography resins for immunoprecipitation (IP) of antibodies from biological solutions [ 11 , 18 , 29 , 30 , 39 ].…”

Section: Introductionmentioning

confidence: 99%

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

Ghaedmohammadi

Ahmadian

2021

Microb Cell Fact

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Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

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Mechanism of antibodies purification by protein A

Cited by 41 publications

References 76 publications

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Interactions between Amino Acids and Zirconia Modified with Ethylenediaminetetra(methylenephosphonic acid): Mechanistic Insights into the Selective Binding of Antibodies

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

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