Novel bioengineering strategies for the recovery and purification of PEGylated lysozyme conjugates: in situ ATPS and affinity chromatography

Hdl Handle:
http://hdl.handle.net/11285/627931
Title:
Novel bioengineering strategies for the recovery and purification of PEGylated lysozyme conjugates: in situ ATPS and affinity chromatography
Issue Date:
2017-07-13
Abstract:

PEGylation is the modification of therapeutic proteins with polyethylene glycol (PEG) with the goal of improving their bioavailability and effectivity in the organism. During the PEGylation process, proteins with different degrees of PEGylation and positional isomers are generated. Numerous chromatographic and non-chromatographic techniques have been used for the purification of the adequate or most active conjugate. However, the obtained yields are still low, representing an interesting engineering challenge to address. Therefore, novel techniques to achieve this must be devised. In the present work, two strategies were explored: in situ aqueous two-phase systems (ATPS) and affinity chromatography.
Aqueous two-phase systems (ATPS) are a promising alternative for recovering modified proteins but this technique has not been tested with complete PEGylation reactions. In this work, lysozyme PEGylation reactions were used as part of the phase-forming chemicals to form in situ ATPS. This was best achieved by adding a 4M ammonium sulphate in a 20 mM Tris-HCl pH 7.0 solution. The phases were separated and analyzed by monolithic chromatography and SDS-PAGE. Results indicate that PEGylated lysozymes (mono- and di-) are mainly fractionated to the top phase (56% and 100% respectively) while native lysozyme was found in the bottom phase (97.7%).
On the other hand, PEG-modified and native lysozyme adsorption to Heparin Sepharose was described by Langmuir isotherms. The affinity of the conjugates decreased with the PEGylation degree with no significant binding of the reactive 20 kDa mPEG to the resin. A method in Heparin Affinity Chromatography (HAC) eluting with NaCl gradient was developed and optimized through Response Surface Methodology for the purification of mono-PEGylated lysozyme with a better yield, purity and productivity than other reported chromatographic modes. A formulated rate model could model and simulate the separation of mono-PEGylated and native lysozymes in HAC. Diverse mass transfer data were obtained from this simulation.
Finally, as an antecedent to the generation of immunosorbents to purify PEGylated proteins, the immobilization conditions of antibodies on NHS Sepharose 4 Fast Flow were
viii
optimized with a factorial design. The antibody isotype showed effects on the coupling efficiency, being improved when isotype G was used.
In conclusion, in situ ATPS and heparin affinity chromatography combined with optimization tools such as design of experiments, modelling and simulation represent new and never reported before techniques in the recovery and purification of PEGylated lysozyme with several advantages with respect to currently used methods.

Keywords:
PEGylated proteins; PEGylation; in situ ATPS; Heparin Affinity Chromatography; simulation; mono-PEGylated lysozyme; optimization; covalent immobilization
Degree Program:
Doctorado en Biotecnología
Advisors:
Marco Antonio Rito Palomares; Karla Patricia Mayolo Deloisa; José Guillermo González Valdez
Committee Member / Sinodal:
Juan A. Asenjo de Leuze; Roberto Parra Saldívar
Degree Level:
Doctor in Biotechnology
School:
School of Engineering and Sciences
Campus Program:
Campus Monterrey
Discipline:
Ingeniería y Ciencias Aplicadas / Engineering & Applied Sciences
Appears in Collections:
Ciencias Exactas

Full metadata record

DC FieldValue Language
dc.contributor.advisorMarco Antonio Rito Palomaresen
dc.contributor.advisorKarla Patricia Mayolo Deloisaen
dc.contributor.advisorJosé Guillermo González Valdezen
dc.creatorMejía, Luisen
dc.date.accessioned2017-10-11T16:03:01Z-
dc.date.available2017-10-11T16:03:01Z-
dc.date.issued2017-07-13-
dc.identifier.urihttp://hdl.handle.net/11285/627931-
dc.description.abstract<p>PEGylation is the modification of therapeutic proteins with polyethylene glycol (PEG) with the goal of improving their bioavailability and effectivity in the organism. During the PEGylation process, proteins with different degrees of PEGylation and positional isomers are generated. Numerous chromatographic and non-chromatographic techniques have been used for the purification of the adequate or most active conjugate. However, the obtained yields are still low, representing an interesting engineering challenge to address. Therefore, novel techniques to achieve this must be devised. In the present work, two strategies were explored: in situ aqueous two-phase systems (ATPS) and affinity chromatography.<br />Aqueous two-phase systems (ATPS) are a promising alternative for recovering modified proteins but this technique has not been tested with complete PEGylation reactions. In this work, lysozyme PEGylation reactions were used as part of the phase-forming chemicals to form in situ ATPS. This was best achieved by adding a 4M ammonium sulphate in a 20 mM Tris-HCl pH 7.0 solution. The phases were separated and analyzed by monolithic chromatography and SDS-PAGE. Results indicate that PEGylated lysozymes (mono- and di-) are mainly fractionated to the top phase (56% and 100% respectively) while native lysozyme was found in the bottom phase (97.7%).<br />On the other hand, PEG-modified and native lysozyme adsorption to Heparin Sepharose was described by Langmuir isotherms. The affinity of the conjugates decreased with the PEGylation degree with no significant binding of the reactive 20 kDa mPEG to the resin. A method in Heparin Affinity Chromatography (HAC) eluting with NaCl gradient was developed and optimized through Response Surface Methodology for the purification of mono-PEGylated lysozyme with a better yield, purity and productivity than other reported chromatographic modes. A formulated rate model could model and simulate the separation of mono-PEGylated and native lysozymes in HAC. Diverse mass transfer data were obtained from this simulation.<br />Finally, as an antecedent to the generation of immunosorbents to purify PEGylated proteins, the immobilization conditions of antibodies on NHS Sepharose 4 Fast Flow were<br />viii<br />optimized with a factorial design. The antibody isotype showed effects on the coupling efficiency, being improved when isotype G was used.<br />In conclusion, in situ ATPS and heparin affinity chromatography combined with optimization tools such as design of experiments, modelling and simulation represent new and never reported before techniques in the recovery and purification of PEGylated lysozyme with several advantages with respect to currently used methods.</p>en
dc.language.isoengen
dc.rightsOpen Accessen
dc.titleNovel bioengineering strategies for the recovery and purification of PEGylated lysozyme conjugates: in situ ATPS and affinity chromatographyen
dc.typeTesis de Doctoradoes
thesis.degree.grantorInstituto Tecnológico y de Estudios Superiores de Monterreyes
thesis.degree.levelDoctor in Biotechnologyen
dc.contributor.committeememberJuan A. Asenjo de Leuzeen
dc.contributor.committeememberRoberto Parra Saldívaren
thesis.degree.disciplineSchool of Engineering and Sciencesen
thesis.degree.nameDoctorado en Biotecnologíaen
dc.subject.keywordPEGylated proteinsen
dc.subject.keywordPEGylationen
dc.subject.keywordin situ ATPSen
dc.subject.keywordHeparin Affinity Chromatographyen
dc.subject.keywordsimulationen
dc.subject.keywordmono-PEGylated lysozymeen
dc.subject.keywordoptimizationen
dc.subject.keywordcovalent immobilizationen
thesis.degree.programCampus Monterreyen
dc.subject.disciplineIngeniería y Ciencias Aplicadas / Engineering & Applied Sciencesen
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