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Peptide Affinity Ligands for the Pur...

Moore, Brandyn David.

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Peptide Affinity Ligands for the Purification of Labile Biologics.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Peptide Affinity Ligands for the Purification of Labile Biologics./
Author:	Moore, Brandyn David.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2023,
Description:	174 p.
Notes:	Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
Contained By:	Dissertations Abstracts International85-06B.
Subject:	Load. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30727232
ISBN:	9798381021523

Peptide Affinity Ligands for the Purification of Labile Biologics.
Moore, Brandyn David.

Peptide Affinity Ligands for the Purification of Labile Biologics. - Ann Arbor : ProQuest Dissertations & Theses, 2023 - 174 p.

Source: Dissertations Abstracts International, Volume: 85-06, Section: B.

Thesis (Ph.D.)--North Carolina State University, 2023.

This item must not be sold to any third party vendors.

Purification is one of the most important and costly steps in bioprocess manufacturing. Chromatography is a very common method of purification where typically, a protein adsorbent is used to bind, then release a target when an acidic pH shift is induced. Protein adsorbents are expensive, and the low pH elution can be harmful to sensitive biologics. This work presents an alternative form of adsorbent: peptides. Peptide's smaller structure allows for milder binding, and therefore, milder elution conditions. The work presented here demonstrates the possibility for a light-induced elution, as well as a mild, basic pH shift elution, to purify a blood factor, stem cells, and exosomes.A combinatorial peptide library was cyclized with an azobenzene linker, which is known for its photo-responsive properties, namely photoisomerizing from trans- to cis- when irradiated with UV light, and back from cis- to trans- in response to blue light. This library was screened for combinatorial regions that bound desired targets in the cis- isomer and released the target when switched back into the trans- isomer, so that blue light could act as an elution stimulus.Azobenzene-cyclized peptide affinity ligands were discovered for the purification of human blood clotting factor VIII. Buffer conditions were optimized to achieve sufficient factor VIII purification exclusively with a light-induced isomerization for elution. It was determined that high NaCl concentrations weakened hydrophobic interactions between the peptide and the protein, which allowed for extremely mild elution conditions. Purified factor VIII was tested for activity, and it was proved that purification resulted in little to no loss in activity. The yield and purity of factor VII exceeded 90% when purified from a CHO cell culture fluid feedstock.Cell surface markers CD38 and FLT3, along with VCAM-1, can be used to fractionate hematopoietic stem cells into their respective stages of differentiation. Azobenzene-cyclized peptide ligands have already been discovered for VCAM-1, and this work demonstrates ligands for the purification of recombinant forms of both CD38 and FLT3. During purification of rCD38 and rFLT3, it was shown that photoisomerization of the azobenzene may be used in conjunction with mild pH shifts to elute these proteins from the resin to achieve high purity (>90%) with sufficient yield (>70%).Exosomes are small extracellular vesicles secreted from all cells for signaling purposes. These small vesicles have potential for a variety of applications, including diagnostics and gene therapy. The result of this work is the discovery of a linear peptide affinity ligand that can purify exosomesuniversally, regardless of the source of these exosomes. Exosomes were purified from over 10 different sources with this ligand, including various cancer cell lines, human plasma, human urine, and HEK293 cell culture fluid. Final tests showed exosomes could be recovered from raw concentrated HEK293 cell culture fluid, to a much greater degree of purity and yield than achievable by standard methods.

ISBN: 9798381021523Subjects--Topical Terms:

3562902
Load.

Peptide Affinity Ligands for the Purification of Labile Biologics.
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500 $a Advisor: Menegatti, Stefano;Daniele, Michael;Crook, Nathan;Hall, Carol.
502 $a Thesis (Ph.D.)--North Carolina State University, 2023.
506 $a This item must not be sold to any third party vendors.
520 $a Purification is one of the most important and costly steps in bioprocess manufacturing. Chromatography is a very common method of purification where typically, a protein adsorbent is used to bind, then release a target when an acidic pH shift is induced. Protein adsorbents are expensive, and the low pH elution can be harmful to sensitive biologics. This work presents an alternative form of adsorbent: peptides. Peptide's smaller structure allows for milder binding, and therefore, milder elution conditions. The work presented here demonstrates the possibility for a light-induced elution, as well as a mild, basic pH shift elution, to purify a blood factor, stem cells, and exosomes.A combinatorial peptide library was cyclized with an azobenzene linker, which is known for its photo-responsive properties, namely photoisomerizing from trans- to cis- when irradiated with UV light, and back from cis- to trans- in response to blue light. This library was screened for combinatorial regions that bound desired targets in the cis- isomer and released the target when switched back into the trans- isomer, so that blue light could act as an elution stimulus.Azobenzene-cyclized peptide affinity ligands were discovered for the purification of human blood clotting factor VIII. Buffer conditions were optimized to achieve sufficient factor VIII purification exclusively with a light-induced isomerization for elution. It was determined that high NaCl concentrations weakened hydrophobic interactions between the peptide and the protein, which allowed for extremely mild elution conditions. Purified factor VIII was tested for activity, and it was proved that purification resulted in little to no loss in activity. The yield and purity of factor VII exceeded 90% when purified from a CHO cell culture fluid feedstock.Cell surface markers CD38 and FLT3, along with VCAM-1, can be used to fractionate hematopoietic stem cells into their respective stages of differentiation. Azobenzene-cyclized peptide ligands have already been discovered for VCAM-1, and this work demonstrates ligands for the purification of recombinant forms of both CD38 and FLT3. During purification of rCD38 and rFLT3, it was shown that photoisomerization of the azobenzene may be used in conjunction with mild pH shifts to elute these proteins from the resin to achieve high purity (>90%) with sufficient yield (>70%).Exosomes are small extracellular vesicles secreted from all cells for signaling purposes. These small vesicles have potential for a variety of applications, including diagnostics and gene therapy. The result of this work is the discovery of a linear peptide affinity ligand that can purify exosomesuniversally, regardless of the source of these exosomes. Exosomes were purified from over 10 different sources with this ligand, including various cancer cell lines, human plasma, human urine, and HEK293 cell culture fluid. Final tests showed exosomes could be recovered from raw concentrated HEK293 cell culture fluid, to a much greater degree of purity and yield than achievable by standard methods.
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