Separation of human Fab fragments by negative chromatography on ω-aminohexyl- and poly(ethyleneimine)-agarose

Carmignotto, Gabriela Pannunzio; Mourão, Cecília Alves; Bueno, Sônia Maria Alves

doi:10.1016/j.procbio.2016.10.013

Cited by 9 publications

(5 citation statements)

References 32 publications

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“…However, the use of surfactants in this early phase of the STEAP1 isolation was discarded to not jeopardize further characterization techniques which, often, require their posterior removal by dialysis or similar methodologies. So far, negative chromatography was already used for the purification of antibodies [ 36 , 37 ], recombinant proteins [ 38 , 39 ] and virus-like particles [ 40 , 41 ] exhibiting better purity and recovery performances than other commonly reported techniques and surpassing the binding capacity limitation of typical chromatographic matrices [ 42 ].…”

Section: Resultsmentioning

confidence: 99%

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

Barroca-Ferreira

Cruz-Vicente

Santos

et al. 2021

IJMS

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Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

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Section: Resultsmentioning

confidence: 99%

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

Barroca-Ferreira

Cruz-Vicente

Santos

et al. 2021

IJMS

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“…The concentration of Fab fragments in the chromatographic fractions was determined by radial immunodiffusion (RID) assay, in accordance with da Silva et al (), Mourão et al () and Carmignotto et al (). A Fab‐specific anti‐human IgG antibody conjugated to peroxidase produced in goat was used (Sigma‐Aldrich, USA).…”

Section: Methodsmentioning

confidence: 63%

“…Aiming at obtaining Fab and Fc fragments, whole human IgG was cleaved with papain following the procedures described by Ternynck and Avrameas (), da Silva, Serracchiani, Miranda, and Bueno (), Mourão, Carmignotto, and Bueno (), and Carmignotto, Mourão, and Bueno () using a 30 mg mL −1 solution of high purity human IgG in 100 mmol L −1 NaP buffer, pH 7.4 containing 40 mmol L −1 EDTA. Then cysteine‐activated papain was added to the IgG solution to achieve a papain/antibody ratio of 1% (w/w).…”

Section: Methodsmentioning

confidence: 99%

“…After SDS–PAGE runs, protein samples were transferred to nitrocellulose membranes in a Mini Transblotting III System (Bio‐Rad, USA) as described by da Silva et al (), Mourão et al () and Carmignotto et al () for the specific detection of whole human IgG and its Fc and Fab fragments. The secondary antibodies used were peroxidase conjugated anti‐human IgG (Fab specific or Fc specific), both produced in goat and purchased from Sigma‐Aldrich (USA).…”

Section: Methodsmentioning

confidence: 99%

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Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments

Pavan

Bresolin

Muzio

et al. 2018

Biomedical Chromatography

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The behavior of human immunoglobulin G (IgG) and antigen-binding fragment (Fab fragment) adsorption onto phospho-L-tyrosine immobilized on agarose (P-Tyr-agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis-Tris, Tris-HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L −1 NaP buffer at pH 6.0. The capture of IgG by the P-Tyragarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L −1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo-second-order model. The adsorption isotherm data were well described by the Langmuir-Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P-Tyr-agarose from digested human IgG in HEPES 25 mmol L −1 buffer at pH 7.0where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.

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“…(2014),Mourão, Carmignotto & Bueno (2016) eCarmignotto et al (2017), com a finalidade de quantificar a hIgG presente nas amostras das etapas cromatográficas da melhor condição de purificação. Inicialmente o gel de agarose foi preparado, o qual O gel foi corado por submersão por 30 minutos em solução de Coomassie brilliant blue R-250 (0,5% m/v), etanol (45% v/v) e ácido acético (45% v/v).…”

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Síntese e caracterização de criogéis de poliacrilamida-quitosana com íons metálicos Cu2+ e Ni2+ imobilizados para a purificação de imunoglobulina G humana por IMAC

Sepúlveda Del Rio Hamacek

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Antibodies or immunoglobulins are amongst the proteins that require the most degree of purity for their applications, such as in the diagnostic and treatment of diseases. Current purification methods, in which allow high yields and degree of purity, for those biomolecules can involve affinity chromatography, a technique that is based on the selective interaction between an immobilized ligand and the product of interest. The utilization of those procedures leads to the reduction of the number of additional steps, costs and processing time when compared to other protein purification methods. This work aimed to synthetize and characterize a monolithic stationary phase with adequate properties to be used in immobilized metal ion affinity chromatography to separate immunoglobulin G (hIgG) from human serum. Different conditions of cryopolimerization of the acrylamide, bisacrylamide, allyl-glycidyl-ether in the presence of chitosan chains and the crosslinking agent glutaraldehyde were tested and the cryogel that proved more suitable was selected. The chelating agent iminodiacetic acid (IDA) was covalently immobilized onto the cryogel, which was named as PAAm-CS-GA-AGE-IDA. Then, the adsorption and purification capacities of hIgG by the cryogel in the presence or not of chelated Cu 2+ and Ni 2+ ions were investigated. In the cryogel with chelated Ni 2+ , the sodium phosphate buffer system at pH 7,0 (25 mmol L -1 ) with imidazole (2 mmol L -1 ) proved to be the most promising condition for providing yield of 55% and purity of 88% for hIgG. The breakthrough curve revealed that the cryogel continued to be selective for hIgG under saturation conditions, resulting in 96% of purity. The kinetics experiments evidenced that the equilibrium time of the adsorption is quickly reached. Finally, adsorption isotherms were built and the parameters of the Langmuir and Langmuir-Freundlich models were adjusted by non-linear regression.The Langmuir-Freundlich model represented well the adsorption experimental data, resulting in the maximum capacity equal to 31,19 mg hIgG/g dry cryogel and a dissociation constant in the order of Kd = 10 -7 mol L -1 . The obtained results proved the potential of the cryogel of polyacrylamide and chitosan with immobilized metal ions to separate and purify hIgG from human serum.

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Separation of human Fab fragments by negative chromatography on ω-aminohexyl- and poly(ethyleneimine)-agarose

Cited by 9 publications

References 32 publications

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments

Síntese e caracterização de criogéis de poliacrilamida-quitosana com íons metálicos Cu2+ e Ni2+ imobilizados para a purificação de imunoglobulina G humana por IMAC

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