Purification of antibodies by affinity chromatography

Huse, Klaus; Böhme, H; Scholz, Gerhard H.

doi:10.1016/s0165-022x(02)00017-9

Cited by 222 publications

(136 citation statements)

References 65 publications

Supporting

Mentioning

110

Contrasting

Unclassified

Order By: Relevance

“…Both aptamers bound to all hIgG subclasses, hIgG1 through hIgG4, but not to other IgGs examined so far, such as those from mouse, rat, rabbit, bovine, chicken, canine, feline, and guinea pig (data not shown). This high selectivity is in sharp contrast to protein A, which shows much less stringent affinity and binds to these IgGs, except for chicken IgG, rat IgG2a, rat IgG2b, and bovine IgG1 (Huse et al 2002).…”

Section: High Specificity Of Apt2 and Apt8 To Human Iggmentioning

confidence: 72%

“…At present, protein A affinity chromatography is the common procedure used in purification of antibodies, because it selectively and efficiently binds antibodies in complex solutions, such as harvested cell culture media (Fahrner et al 2001;Ghose et al 2005). Protein A is a natural product of Staphylococcus aureus; it binds the Fc portion of a variety of mammalian IgG molecules (Huse et al 2002).…”

Section: Introductionmentioning

confidence: 99%

“…To overcome the disadvantages, several synthetic ligands have been proposed as replacements for protein A in the affinity purification of antibodies; these include the use of a thiophilic ligand (Porath et al 1985), histidyl ligand (Elkak and Vjiayalakshmi 1991), Avid A1 (Ngo and Khatter 1990), or peptides or nonpeptides designed to mimic protein A (Fassina et al 2001; for review, see Huse et al 2002). However, to our knowledge none of these have as yet become protein A alternatives at the manufacturing level.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

Miyakawa¹,

Nomura²,

Sakamoto³

et al. 2008

RNA

109

107

View full text Add to dashboard Cite

Potential applications for functional RNAs are rapidly expanding, not only to address functions based on primary nucleotide sequences, but also by RNA aptamer, which can suppress the activity of any target molecule. Aptamers are short DNA or RNA folded molecules that can be selected in vitro on the basis of their high affinity for a target molecule. Here, we demonstrate the ability of RNA aptamers to recognize-and bind to-human IgG with high specificity and affinity. An optimized 23-nucleotide aptamer, Apt8-2, was prepared, and was shown to bind to the Fc domain of human IgG, but not to other IgG's, with high affinity. Apt8-2 was observed to compete with protein A, but not with the Fcg receptor, for IgG binding. NMR chemical-shift analyses localized the aptamer-binding sites on the Fc subdomain, which partially overlaps the protein A binding site but not the Fcg receptor binding site. The tertiary structures of the predicted recognition sites on the Fc domain differ significantly between human IgG and other species of IgGs; this, in part, accounts for the high specificity of the selected aptamer. Apt8-2 can therefore be used as a protein A alternative for affinity purification of human IgG and therapeutic antibodies. Using Apt8-2 would have several potential advantages, raising the possibility of developing new applications based on aptamer design.

show abstract

Section: High Specificity Of Apt2 and Apt8 To Human Iggmentioning

confidence: 72%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

Miyakawa¹,

Nomura²,

Sakamoto³

et al. 2008

RNA

109

107

View full text Add to dashboard Cite

show abstract

“…Tradicionalmente, a purificação dessas biomoléculas é realizada por meio da técnica de cromatografia de afinidade, empregando como ligantes a proteína A ou a proteína G imobilizadas (Huse et al, 2002). No entanto, esses ligantes apresentam algumas desvantagens, tais como o elevado custo, o risco de desnaturação do anticorpo e de desprendimento do ligante da matriz em decorrência das condições de eluição a baixos valores de pH e o risco da inativação do ligante após sucessivos ciclos de purificação (Ayyar et al, 2012).…”

Section: Nos úLtimos Anos Vários Fragmentos De Igg Foram Aprovados Punclassified

PURIFICAÇÃO DE FRAGMENTOS Fab POR IMAC EM AGAROSE-CM-Asp-Co(II)

Mourão¹,

Bueno²

2015

Anais Do XX Congresso Brasileiro De Engenharia Química

View full text Add to dashboard Cite

RESUMO -As imunoglobulinas G (IgG) e seus fragmentos Fab, F(ab)' 2 e Fv apresentam aplicações relevantes na área médica e de diagnóstico. O elevado custo de purificação dessas biomoléculas por técnicas de afinidade justifica a investigação de alternativas de baixo custo para a sua purificação. O objetivo deste trabalho é avaliar a eficácia do quelato CM-Asp-Co(II) imobilizado em agarose na purificação dos fragmentos Fab de IgG humana, a partir de uma solução de IgG clivada pela enzima papaína. O efeito do sistema tamponante e do cloreto de sódio foram avaliados. O tampão Hepes na ausência de sal, permitiu obter os fragmentos Fab com pureza superior a 90%. Esse tampão possibilitou a maior recuperação seletiva dos fragmentos, em comparação com o tampão Hepes contendo NaCl e com os tampões, Tris-HCl e fosfato de sódio. Os resultados evidenciaram a potencialidade desse ligante para purificação de fragmentos Fab. INTRODUÇÃOOs fragmentos Fab, F(ab)' 2 e Fv obtidos da clivagem da IgG ou por meio da tecnologia do DNA recombinante, são proteínas que apresentam aplicações relevantes em terapias e diagnósticos. Tais biomoléculas são caracterizadas por possuírem massa molar e tempo de meia-vida inferiores ao da IgG intacta, consequentemente apresentam maior facilidade de penetração nos tecidos alvo e menor tempo de depuração plasmática (Chames et al., 2009). Nos últimos anos, vários fragmentos de IgG foram aprovados pelo FDA (Food and DrugAdministration) e têm sido empregados em diagnósticos por imagem in vivo, nas terapias de doenças degenerativas e cânceres (Hansel et al., 2010). A aplicação dos fragmentos de anticorpos nesses segmentos representa, aproximadamente, 6 bilhões de dólares por ano (Holliger e Hudson, 2005).Para serem empregados nessas aplicações é requerido um elevado grau de pureza da IgG e de seus fragmentos. Tradicionalmente, a purificação dessas biomoléculas é realizada por meio da técnica de cromatografia de afinidade, empregando como ligantes a proteína A ou a proteína G imobilizadas (Huse et al., 2002). No entanto, esses ligantes apresentam algumas desvantagens, tais como o elevado custo, o risco de desnaturação do anticorpo e de desprendimento do ligante da matriz em decorrência das condições de eluição a baixos valores de pH e o risco da inativação do ligante após sucessivos ciclos de purificação (Ayyar et al., 2012). Diante desses inconvenientes, estudos têm sido desenvolvidos visando o emprego de técnicas alternativas à cromatografia de afinidade (Coleman e Mahler, 2003; Yu e Ghosh, 2010) ou de ligantes substitutos das proteínas A e G (Khoury e Lowe, Área temática: Processos Biotecnológicos 1

show abstract

“…Although several Protein-A Mimetic peptide ligands have been synthesized, screened and evaluated for their suitability in chromatography process development (Fassina, 2000;Roque et al, 2004;Yang et al, 2005), only a few have been extensively studied. For example, PAM peptide TG19318 (Fassina et al, 1998), a tetrameric peptide ligand, was successfully investigated for the isolation of various classes of human Igs (IgG, IgA, IgE, IgM) (Huse et al, 2002) from human serum and bacterial cell culture broths. Short linear hexameric peptide ligands HWRGWV, HYFKFD and HFRRHL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 were identified and developed by Carbonell's group through screening of combinatorial libraries on a solid resin and its selectivity towards human Igs through the Fc region was characterised (Yang et al, 2005(Yang et al, , 2009.…”

Section: Introductionmentioning

confidence: 99%

Application of peptide chromatography for the isolation of antibodies from bovine skim milk, acid whey and colostrum

Billakanti

Fee

Naik

et al. 2014

Food and Bioproducts Processing

View full text Add to dashboard Cite

Protein A mimetic peptide ligands have several benefits over conventional Protein A/G ligands, namely that they are small in size, have low production costs, are stable over a wide range of pH values and can withstand cleaning by harsh sanitization agents such as sodium hydroxide. In this paper, a hexamer peptide (HWRGWV) affinity matrix was used for the isolation of bovine immunoglobulins from various dairy streams (skim milk, acid whey and colostrum). Bound immunoglobulins were recovered in elution buffer (0.2 M sodium acetate buffer, pH 4.0) fractions with a purity of >85% in a single step. The peptide resin has achieved a maximum equilibrium adsorption capacity of 23±0.58 mg.mL-1 of resin for bovine IgG and had a dynamic binding capacity of 11.8±0.03 mg.mL-1 at residence time of 2 min. These results suggest that the hexamer peptide chromatography could potentially be used for the selective purification of bovine immunoglobulins from dairy streams. This method has promise as an alternative to conventional Protein A/G chromatography for direct capture of immunoglobulins from streams containing relatively high immunoglobulin concentrations such as colostrum, transgenic or hyper-immune milk. In the abstract, equilibrium adsorption capacity should appear with the associated error (in the previous submission this error appear).Decision: Authors are accepted the above and reviewed as suggested (23±0.58). Comment2.Page 12 line 10 "However, for experiments with standard bIgG at 1 mg mL-1 and 9 mg mL-1 were overloaded" The idea to answer comment 8, reviewer 1 is there but the sentence should better introduced in the text.Decision: Authors are accepted the above and reviewed as suggested in the draft. "However, for experiments with standard bIgG at 1 mg.mL -1 and 9 mg.mL -1 were overloaded since column reached its dynamic capacity approximately at 5.5 mL for 1 mg.mL -1 bIgG and 1.4 mL for 9 mg.mL -1 bIgG. Due to this, % of IgG bound data presented in table 1.1 for these experiments may not reflect the actual numbers". Thank you.Yours sincerely, Jagan M Billakanti, PhD. Cover LetterRevision 2 Reviewer #1 Comment1. In the abstract, equilibrium adsorption capacity should appear with the associated error (in the previous submission this error appear).Decision: Authors are accepted the above and reviewed as suggested (23±0.58) Comment2. Page 12 line 10 "However, for experiments with standard bIgG at 1 mg mL-1 and 9 mg mL-1 were overloaded" The idea to answer comment 8, reviewer 1 is there but the sentence should better introduced in the text.Decision: Authors are accepted the above and reviewed as suggested in the draft."However, for experiments with standard bIgG at 1 mg.mL -1 and 9 mg.mL -1 were overloaded since column reached its dynamic capacity approximately at 5.5 mL for 1 mg.mL -1 bIgG and 1.4 mL for 9 mg.mL -1 bIgG. Due to this, % of IgG bound data presented in table 1.1 for these experiments may not reflect the actual numbers". Detailed Response to ReviewersHighlights  HWRGWV peptide resin showed an equ...

show abstract

Purification of antibodies by affinity chromatography

Cited by 222 publications

References 65 publications

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G

PURIFICAÇÃO DE FRAGMENTOS Fab POR IMAC EM AGAROSE-CM-Asp-Co(II)

Application of peptide chromatography for the isolation of antibodies from bovine skim milk, acid whey and colostrum

Contact Info

Product

Resources

About