Protein purification by affinity precipitation

Hilbrig, Frank; Freitag, Ruth

doi:10.1016/s1570-0232(03)00081-3

Cited by 105 publications

(38 citation statements)

References 60 publications

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“…Ethanol was added to the samples purified until 50-80% (v/v) of fractional concentrations were obtained. This mixture was agitated and centrifuged at 4000rpm and 4°C for 15min (Cortez and Pessoa Jr., 1999;Hilbring and Freitag, 2003). Total protein and enzymatic activity were determined in the supernatant (samples showed no precipitate) by Bradford and DNS methods.…”

Section: Amylases Purificationmentioning

confidence: 99%

Production and characterization of amylases from Zea mays malt

Biazus

Souza

Marquez

et al. 2009

Braz. arch. biol. technol.

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Section: Amylases Purificationmentioning

confidence: 99%

Production and characterization of amylases from Zea mays malt

Biazus

Souza

Marquez

et al. 2009

Braz. arch. biol. technol.

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“…These range from salting-out the antibody with ammonium sulfate (Venkiteshwaran et al, 2008) to affinity precipitation with smart polymers or affinity macroligands. Affinity macroligands consist of polymers onto which affinity ligand(s) are conjugated (Hilbrig and Freitag, 2003;Mattiasson et al, 1998a, b). The polymer can vary from elastin like polypeptides that precipitate with a change in temperature (Kim et al, 2005) to copolymers of methacrylic acid and methyl methacrylate (Eudragit S-100) that precipitate with changing pH (Taipa et al, 1998(Taipa et al, , 2001.…”

Section: Introductionmentioning

confidence: 99%

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

McDonald

Victa

Carter-Franklin

et al. 2008

Biotech & Bioengineering

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We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody-polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to < 1 microg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody.

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“…An important aspect of this technique is that the enzyme usually retains its tertiary structure and its catalytic activity. Even more, usually it is more stable in the presence of the polyelectrolyte [9,10].…”

Section: Bioseparation Of Proteins From a Complex Mixturementioning

confidence: 99%