Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems

Azevedo, Ana M.; Gomes, G. G.; Rosa, P.A.J.; Ferreira, Isabel; Pisco, Angela M.M.O.; Aires-Barros, M. Raquel

doi:10.1016/j.seppur.2007.12.010

Cited by 202 publications

(103 citation statements)

References 21 publications

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“…The observed salting-out effect of the antibody is consistent with observations in other published work. 16,23,[25][26][27] It was also noticed by Aires-Barros group and others that the citrate anion demonstrated more prominent salting-out effect than the phosphate anions 24,37 and NaCl, as a neutral salt, can increase the difference in hydrophobicity between two phases. 38 In solution, a stable protein (i.e., antibody) normally folds in a way that its hydrophobic core is buried inside while hydrophilic residues face outside and interact with water molecules through hydrogen bonding.…”

Section: Driving Force For Antibody Partitioningmentioning

confidence: 84%

“…Purified protein was selected for this initial investigation because this would allow rapid titer determination using A280 absorbance without a concern for background interference that would exist if HCCF were used. Factors and ranges for the resolution 2 5À1 V two-level factorial design were selected based upon the established phase diagrams 16,26 and expected manufacturing constraints ( Table 2).…”

Section: Screening Experimental Designmentioning

confidence: 99%

“…Early work focused on extraction of mAb from hybridoma cell culture supernatant using a PEG-phosphate system. 22,23 Recently, the following extraction systems have been used to purify either hybridoma or Chinese Hamster Ovary (CHO) cell-derived mAb: PEG-phosphate, 24,25 PEGcitrate, 26,27 PEG-dextran, 28,29 and PEG-dextran using modified PEG molecules with different functional groups. 30,31 Although antibody partition was demonstrated in these studies, data pertaining to the partitioning of impurities, such as host cell protein (HCP), was limited.…”

Section: Introductionmentioning

confidence: 99%

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Downstream antibody purification using aqueous two‐phase extraction

Mao

Rogers

Westoby

et al. 2010

Biotechnology Progress

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The extraction of antibodies using a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6-fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (aggregates and low molecular product fragments) from ∼40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode.

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Section: Driving Force For Antibody Partitioningmentioning

confidence: 84%

Section: Screening Experimental Designmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Downstream antibody purification using aqueous two‐phase extraction

Mao

Rogers

Westoby

et al. 2010

Biotechnology Progress

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“…It is one of low cost and high-efficient purification methods (Asenjo and Andrews 2011). Recently, many enzymes (α-amylase, α-galactosidase, lipase, proteinase), recombinant proteins, and antibody have been purified using this method (Azevedo et al 2009;Porfiri et al 2011;Gu et al 2012;Loc et al 2013;Ramakrishnan et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Liu

Guo

et al. 2018

Bioresour. Bioprocess.

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Background: Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.Results: An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0-7.0 at 55 °C for 60 min. The enzyme exhibited K m , V max , K cat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 10 3 /S, respectively. The addition of 10 mM of Mg 2+ , Mn 2+ , or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity. Conclusions:An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.

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“…For the purifi cation of termolabile and sensitive proteins, such as hSCOMT, a good alternative to AS can be sodium citrate (SC), a biodegradable, anti-chaotropic ion described as a powerful protein stabilizer (Bottomley and Tew, 2000) and used in the isola-tion of several biomolecules, such as antibodies (Azevedo et al, 2009) and small proteins (Sousa et al, 2008). Also, the temperature of the chromatographic system is a crucial factor in improving HIC selectivity, despite less progress in elucidating the eff ect of this parameter on COMT binding/elution studies.…”

Section: Introductionmentioning

confidence: 99%

Assessment of COMT isolation by HIC using a dual salt system and low temperature

Nunes

Bonifácio

Queiroz

et al. 2010

Biomedical Chromatography

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Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.

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Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems

Cited by 202 publications

References 21 publications

Downstream antibody purification using aqueous two‐phase extraction

Downstream antibody purification using aqueous two‐phase extraction

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Assessment of COMT isolation by HIC using a dual salt system and low temperature

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