Antibodies and Genetically Engineered Related Molecules: Production and Purification

Roque, Ana C. A.; Lowe, Christopher R.; Taipa, M. A.

doi:10.1021/bp030070k

Cited by 328 publications

(216 citation statements)

References 175 publications

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“…Purification of pharmaceutical and other high-value proteins is an ongoing concern for biotechnologists, because commercial downstream processes used today are extremely complex and costly, accounting for as much as 50-80% of total manufacturing costs (25). In particular, the cost of protein A-based immunoadsorbent, a standard reagent used for industrial purification of monoclonal antibodies, is so high that the columns have to be reused up to 50 times, resulting in significant costs due to multiple cleanings and revalidation of the columns.…”

Section: Discussionmentioning

confidence: 99%

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

Werner¹,

Marillonnet²,

Hause³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

122

123

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Earlier attempts to express peptides longer than 20 aa on the surface of tobamoviruses such as tobacco mosaic virus have failed. Surprisingly, we found that a functional fragment of protein A (133 aa) can be displayed on the surface of a tobamovirus as a Cterminal fusion to the coat protein via a 15-aa linker. The macromolecular nature of these nanoparticles allowed the design of a simple protocol for purification of mAbs with a recovery yield of 50% and >90% product purity. The extremely dense packing of protein A on the nanoparticles (>2,100 copies per viral particle) results in an immunoadsorbent with a binding capacity of 2 g mAb per g. This characteristic, combined with the high level of expression of the nanoparticles (>3 g adsorbent per kg of leaf biomass), provides a very inexpensive self-assembling matrix that could meet the criteria for a single-use industrial immunoadsorbent for antibody purification.antibody purification ͉ coat protein fusion ͉ viral particle

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Section: Discussionmentioning

confidence: 99%

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

Werner¹,

Marillonnet²,

Hause³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

122

123

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“…Small ligands such as peptides have potential advantages in chromatography processes because they can be more stable and less immunogenic than protein ligands, can be constructed with a wide variety of bio-specificity and their production cost is low (Fassina et al, 1996;Yang et al, 2005). 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.…”

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

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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...

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“…Novel affinity-based separations have emerged from the development of synthetic ligands including peptides obtained by combinatorial libraries and artificial ligands generated by de novo process designs (Roque et al, 2004(Roque et al, , 2007, although so far with limited applicability by big pharma companies. Nonchromatographic alternatives including membrane chromatography, tangential flow filtration, high gradient magnetic fishing, aqueous two-phase extraction, precipitation, and crystallization have also been described (Przybycien et al, 2004;Low et al, 2007;Azevedo et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

Azevedo

Gomes

Borlido

et al. 2010

J of Molecular Recognition

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In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.

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Antibodies and Genetically Engineered Related Molecules: Production and Purification

Cited by 328 publications

References 175 publications

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

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

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

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