Extraction of Human IgG in Thermo-Responsive Aqueous Two-Phase Systems: Assessment of Structural Stability by Circular Dichroism

Borlido, Luis; Azevedo, Ana M.; Aires-Barros, M. Raquel

doi:10.1080/01496395.2010.507441

Cited by 5 publications

(4 citation statements)

References 13 publications

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“…In previous studies reporting to the use of polymer-based ABS, the extraction efficiency of IgG in a single-step was found to be < 90% (Rosa et al, 2007; 2009; Azevedo et al, 2007; 2008). In some works extraction efficiencies higher than 90% were obtained, although in these cases multiple steps are required (Rosa et al, 2013; Ferreira et al, 2008; Borlido et al, 2010). In the systems investigated in this work, the addition of 5 wt% of ILs as adjuvants in polymer-salt ABS leads to 100% of extraction efficiency of IgG to the polymer-rich phase in a single-step.…”

Section: Resultsmentioning

confidence: 99%

Improving the extraction and purification of immunoglobulin G by the use of ionic liquids as adjuvants in aqueous biphasic systems

Ferreira

Faustino

Mondal

et al. 2016

Journal of Biotechnology

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Immunoglobulins G (IgG) could become widespread biopharmaceuticals if cost-efficient processes for their extraction and purification are available. In this work, aqueous biphasic systems (ABS) composed of polyethylene glycols and a buffered salt, and with ionic liquids (ILs) as adjuvants, have been studied as alternative extraction and purification platforms of IgG from a rabbit serum source. Eleven ILs were investigated to provide insights on the chemical features which maximize the IgG partitioning. It is shown that in polymer-salt systems pure IgG preferentially partitions to the polymer-rich phase; yet, the complete extraction was never attained. Remarkably, after the addition of 5wt% of adequate ILs to polymer-salt ABS, the complete extraction of pure IgG in a single-step was accomplished. The best systems and conditions were then applied to the extraction and purification of IgG directly from rabbit serum samples. The complete extraction of IgG in a single-step was maintained while its purity in the polymer-rich phase was enhanced by ca. 37% as compared to the IL-free ABS. The antibody stability was also evaluated revealing that appropriate ILs are able to maintain the IgG stability and can be used as phase-forming components of ABS when envisaging the purification of high-cost biopharmaceuticals.

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Section: Resultsmentioning

confidence: 99%

Improving the extraction and purification of immunoglobulin G by the use of ionic liquids as adjuvants in aqueous biphasic systems

Ferreira

Faustino

Mondal

et al. 2016

Journal of Biotechnology

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“…With the destruction of the secondary structure, the chirality of the protein gradually weakens, and the difference in the absorption of polarized light decreases. Signals in the far-UV CD region (216 nm) were monitored as a function of temperature that was ramping from 25 to 80 °C with a heating rate of 0.5 °C/min . The data interval was 1 °C and every sampling point waited for 120 s to reach equilibrium.…”

Section: Methodsmentioning

confidence: 99%

“…Signals in the far-UV CD region (216 nm) were monitored as a function of temperature that was ramping from 25 to 80 °C with a heating rate of 0.5 °C/min. 38 The data interval was 1 °C and every sampling point waited for 120 s to reach equilibrium. The unfolding fraction, f u , was calculated using…”

Section: ■ Introductionmentioning

confidence: 99%

Creatinase: Using Increased Entropy to Improve the Activity and Thermostability

et al. 2023

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Improving protein thermostability in mutagenesis-based enzyme engineering was often achieved by enhancing interresidue interactions via mutation to increase the enthalpy penalty of unfolding. However, this approach may trade off the functional activity due to the loss of structural flexibility of the biomolecule. Here, by performing X-ray crystallography, enzymatic kinetic experiments, neutron scattering, and thermodynamical measurements, we compared the structures, catalytic behaviors, dynamics, and thermostability between a wild-type creatinase and its four-point mutant. We found that the mutant is an entropy-driven thermostable protein with higher structural flexibility, i.e., higher conformational entropy, in the folded state compared to the wild type. The increased conformational entropy of the mutant in the folded state can reduce the entropy gain during unfolding and thus renders it greater thermostability. Moreover, the increased structural flexibility, particularly around the catalytic site, can broaden the mutant's working temperature range and considerably improve its activity at ambient conditions, which is crucial for its application in diagnosing kidney diseases. Complementary all-atom molecular dynamics simulations indicated that the four mutations replaced several of the strong interresidue interactions (electrostatic interactions and hydrogen bonds) with weak hydrophobic interactions. These substitutions not only release the structural flexibility to promote the thermostability and enzymatic activity of the protein but they also preserve the protein structure from collapsing. Our findings may pave a route for the entropy-driven strategy to design proteins with high thermostability and activity.

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“…Table 7 presents a comparison between the performance of different ATPS in terms of recovery yields and purity levels studied by Aires-Barros and co-workers [17, 155, 163, 178 -191]. ATPS based on PEG/phosphate [163, 178 -181, 190], PEG/sodium citrate [182,183], PEG/dextran [17, 155, 184 -188, 191], and UCON/dextran [189], have been used for the extraction and purification of antibodies from real matrices. [191] Legend: ATPS -aqueous two-phase system; AEX -anion exchange chromatography; AM -artificial mixture; CEX -cation exchange chromatography; CHO -chinese hamster ovary cells; GA -diglutaric acid (COOH); GA-APBA-MP -gum arabic coated particles modified with aminophenyl boronic acid; HIChydrophobic interaction chromatography; HYB -hybridoma cells; LYTAG-Z -choline binding polypeptide tag fused to the synthetic antibody binding Z-domain; PEG -polyethylene glycol; PER.C6 -human embryonic cells derived from retinoblasts; PF -purification factor; SEC -size-exclusion chromatography; TEG-GA -triethylene glycol diglutaric acid; UCON -ethylene oxide/propylene oxide.…”

Section: Aqueous Two-phase Systems (Atps)mentioning

confidence: 99%

Monoclonal Antibodies - Addressing the Challenges on the Manufacturing Processing of an Advanced Class of Therapeutic Agents

Capela¹,

Aires‐Barros²,

Freire³

et al. 2017

Frontiers in Clinical Drug Research: Anti-Infectives Volume 4

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Monoclonal antibodies (mAbs) were firstly described by Köhler and Milstein in 1975, and their potential as powerful therapeutic agents recognized in the following years. Currently, the US Food and Drug Administration (FDA) has already approved 56 monoclonal antibodies for the treatment of several diseases, including cancer (e.g. breast cancer, leukemia and prostate cancer), auto-immune disorders (rheumatoid arthritis and Crohn's disease), asthma, and cardiovascular and infectious diseases. Despite their advantages and therapeutic potential, the cost of manufacturing these biopharmaceuticals with high quality and purity level is still extremely high due to the absence of current cost-effective extraction/purification methods, and which has also impaired their widespread application as recurrent therapeutic agents. The upstream processing of mAbs has gone through several improvements in recent years, by using alternative expression systems or by optimizing the medium formulations and feeding strategies. On the contrary, the downstream processing is considered the bottleneck in the manufacturing of mAbs for therapeutic purposes at reliable costs, representing up to 80% of their total production costs-which is a frontier in clinical drug research. In the past years, several chromatographic and non-chromatographic alternatives have been explored for this purpose, resulting in the development of efficient platforms for the purification of mAbs, that are overviewed and discussed in this chapter. In summary, this chapter provides a vision on the current state of the art of the biopharmaceuticals market, on the production and use of mAbs as valuable therapeutic agents, including their use for the treatment of infectious diseases, while summarizing the mAbs-based products already approved by regulatory agencies. New insights concerning the development of new and alternative platforms or the extraction

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Extraction of Human IgG in Thermo-Responsive Aqueous Two-Phase Systems: Assessment of Structural Stability by Circular Dichroism

Cited by 5 publications

References 13 publications

Improving the extraction and purification of immunoglobulin G by the use of ionic liquids as adjuvants in aqueous biphasic systems

Improving the extraction and purification of immunoglobulin G by the use of ionic liquids as adjuvants in aqueous biphasic systems

Creatinase: Using Increased Entropy to Improve the Activity and Thermostability

Monoclonal Antibodies - Addressing the Challenges on the Manufacturing Processing of an Advanced Class of Therapeutic Agents

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