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M. Raquel Aires-barros - One of the best experts on this subject based on the ideXlab platform.
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Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.
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Partitioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. © 2007 Elsevier B.V. All rights reserved.
Ana M. Azevedo - One of the best experts on this subject based on the ideXlab platform.
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Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.
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Partitioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. © 2007 Elsevier B.V. All rights reserved.
Bernhard O Palsson - One of the best experts on this subject based on the ideXlab platform.
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growth metabolic and antibody production kinetics of Hybridoma Cell Culture 2 effects of serum concentration dissolved oxygen concentration and medium ph in a batch reactor
Biotechnology Progress, 1991Co-Authors: Sadettin S Ozturk, Bernhard O PalssonAbstract:: The effects of serum, dissolved oxygen (DO) concentration, and medium pH on Hybridoma Cell physiology were examined in a controlled batch bioreactor using a murine Hybridoma Cell line (167.4G5.3). The effect of serum was also studied for a second murine Hybridoma Cell line (S3H5/gamma 2bA). Cell growth, viability, Cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and Cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable Cell numbers obtained.
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growth metabolic and antibody production kinetics of Hybridoma Cell Culture 1 analysis of data from controlled batch reactors
Biotechnology Progress, 1991Co-Authors: Sadettin S Ozturk, Bernhard O PalssonAbstract:: A mouse-mouse Hybridoma Cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of Cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The Cell-specific kinetic parameters (growth and metabolic rates) as well as Cell size were constant throughout the exponential phase. IntraCellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The Cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intraCellular antibody content of the Cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.
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simplified method of making alginate polylysine microcapsules for Hybridoma Cell Culture using rpmi 1640 medium
Biotechnology Techniques, 1990Co-Authors: Bernhard O PalssonAbstract:The method of making alginate-poly-L-lysine microcapsules for Hybridoma Cell Culture can be simplified by cultivating the Cells in RPMI 1640 medium. Phosphate concentration in RPMI 1640 medium is sufficiently high to dissolve the alginate gel and, thereby, can be used to eliminate the step of citrate buffer treatment required for reliquefying the interior alginate gel.
Ana Margarida Sintra Pisco - One of the best experts on this subject based on the ideXlab platform.
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Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.
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Partitioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. © 2007 Elsevier B.V. All rights reserved.
A. Gabriela Gomes - One of the best experts on this subject based on the ideXlab platform.
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Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96.
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Partitioning of human antibodies in polyethylene glycol-sodium citrate aqueous two-phase systems
Separation and Purification Technology, 2009Co-Authors: Ana M. Azevedo, A. Gabriela Gomes, Paula A.j. Rosa, I. Filipa Ferreira, Ana Margarida Sintra Pisco, M. Raquel Aires-barrosAbstract:Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a Hybridoma Cell Culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a Hybridoma Cell Culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. © 2007 Elsevier B.V. All rights reserved.
