Purification of Antibodies From Transgenic Plants

Nikolov, Z̆ivko L.; Regan, Jeffrey T.; Dickey, Lynn F.; Woodard, Susan L.

doi:10.1002/9780470444894.ch19

Cited by 12 publications

(10 citation statements)

References 70 publications

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“…This result is in agreement with Mayani et al [64], who showed that the addition of salt at a concentration of 450 mM to the extraction buffer can improve the recovery of recombinant protein in N. benthamiana. The protein extraction process is as essential step of the recovery process because it tailors the total extract volume, concentrations and purity of the recombinant protein as well as the isolation of the desired protein from impurities or/and contaminations that should be removed during the purification process [65,66]. Our data demonstrate that protein recovery increases after salt treatment due to the combination of a reduction in electrostatic interactions between the recombinant protein and plant protein components, such as cellulose that carry a negative charge, and/or increases in osmotic pressure in the extraction buffer, which would result in the dehydration of tissue components [64].…”

Section: Discussionmentioning

confidence: 99%

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

Habibi

Soccol

O’Keefe

et al. 2018

Process Biochemistry

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Section: Discussionmentioning

confidence: 99%

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

Habibi

Soccol

O’Keefe

et al. 2018

Process Biochemistry

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“…5 The extracts of Lemna, tobacco, and other leafy plants can be complex and contain a number of water-soluble nonprotein impurities including phenolics, amino acids, organic acids, carbohydrates, pectin, and nucleic acids. [6][7][8] Phenolic compounds are a potential concern in the production of protein therapeutics such as mAb for two reasons. First, they can interact with proteins through a number of mechanisms including hydrogen bonding, oxidative coupling, and ionic and/or hydrophobic interactions.…”

Section: Introductionmentioning

confidence: 99%

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Barros

Woodard

Nikolov

2011

Biotechnology Progress

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Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ∼ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.

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“…Plants also allow production on a larger scale than is feasible with culturebased systems. mAb or mAb derivatives such as IgG, antibody fragments, and Fc fusion proteins have been successfully expressed in a number of plant host systems (Fischer et al, 1999;Ma et al, 2003;Nikolov et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

“…Plant tissue and leafy green tissue, in particular, present challenges in developing downstream purification processes (Nikolov and Woodard, 2004;Nikolov et al, 2009;Twyman et al, 2003). Plant extracts can contain fibrous tissue that must be removed before subsequent purification steps.…”

Section: Introductionmentioning

confidence: 99%

“…Reports of mAb purification from plant tissue allude to the need for a pretreatment step in order to obtain good recovery on Protein A (Platis and Labrou, 2006;Valdes et al, 2003). Acidification of plant extracts is an easy method which allows the partial removal of host protein (Cox et al, 2006;Nikolov et al, 2009). Aqueous two-phase partitioning has also been used to reduce the amount of phenolics, alkaloids, and pigments (Miller et al, 2004;Platis and Labrou, 2006) in leaf extracts.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development

Woodard

Wilken

Barros

et al. 2009

Biotech & Bioengineering

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Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)-expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin-Ciocalteu assay and by profiling on RP-HPLC. The Folin-Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP-HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA-402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts.

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Purification of Antibodies From Transgenic Plants

Cited by 12 publications

References 70 publications

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development

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