scholarly journals Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

2021 ◽  
Author(s):  
Matthew J McNulty ◽  
Anton Schwartz ◽  
Jesse Delzio ◽  
Kalimuthu Karuppanan ◽  
Aaron Jacobson ◽  
...  
Keyword(s):  
Protein Purification ◽  
Magnetic Separation ◽  
Magnetic Particles ◽  
Binding Capacity ◽  
Therapeutic Protein ◽  
Vector System ◽  
High Yield ◽  
Affinity Capture ◽  
Processing Scheme ◽  
Industry Standards

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via crop cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity of current industry standards by an order of magnitude.

scholarly journals Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Vol 2 (2) ◽  
pp. 35 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

scholarly journals Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

2006 ◽  
Vol 71 (2) ◽  
pp. 135-141 ◽  
Author(s):  
Marija Mojsin ◽  
Jelena Djurovic ◽  
Isidora Petrovic ◽  
Aleksandar Krstic ◽  
Danijela Drakulic ◽  
...  
Keyword(s):  
Dna Binding ◽  
Magnetic Separation ◽  
Magnetic Particles ◽  
Binding Proteins ◽  
Rapid Identification ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
High Yield ◽  
Dna Binding Activity ◽  
Putative Binding Site

In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box) within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

scholarly journals Preactivation Crosslinking – An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

2021 ◽  
Author(s):  
Yasmin Kaveh-Baghbaderani ◽  
Raphaela Allgayer ◽  
Sebastian Patrick Schwaminger ◽  
Paula Fraga-García ◽  
Sonja Berensmeier
Keyword(s):  
Iron Oxide ◽  
Magnetic Separation ◽  
Iron Oxide Nanoparticles ◽  
Binding Capacity ◽  
Protein A ◽  
Oxide Nanoparticles ◽  
High Binding ◽  
A Domains

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

2013 ◽  
Vol 30 (1) ◽  
pp. 108-112 ◽  
Author(s):  
Justin T. McCue ◽  
Keith Selvitelli ◽  
Doug Cecchini ◽  
Rhonda Brown
Keyword(s):  
Protein Purification ◽  
Therapeutic Protein ◽  
Virus Inactivation ◽  
Enveloped Virus

scholarly journals Affinity Membranes and Monoliths for Protein Purification

Membranes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 1 ◽  
Author(s):  
Eleonora Lalli ◽  
Jouciane S. Silva ◽  
Cristiana Boi ◽  
Giulio C. Sarti
Keyword(s):  
Binding Capacity ◽  
Stationary Phases ◽  
Packed Column ◽  
Downstream Processing ◽  
Regenerated Cellulose ◽  
Affinity Capture ◽  
Scale Characterization ◽  
Set Up ◽  
Cellulose Membranes ◽  
Chromatographic Media

Affinity capture represents an important step in downstream processing of proteins and it is conventionally performed through a chromatographic process. The performance of this step highly depends on the type of matrix employed. In particular, resin beads and convective materials, such as membranes and monoliths, are the commonly available supports. The present work deals with non-competitive binding of bovine serum albumin (BSA) on different chromatographic media functionalized with Cibacron Blue F3GA (CB). The aim is to set up the development of the purification process starting from the lab-scale characterization of a commercially available CB resin, regenerated cellulose membranes and polymeric monoliths, functionalized with CB to identify the best option. The performance of the three different chromatographic media is evaluated in terms of BSA binding capacity and productivity. The experimental investigation shows promising results for regenerated cellulose membranes and monoliths, whose performance are comparable with those of the packed column tested. It was demonstrated that the capacity of convective stationary phases does not depend on flow rate, in the range investigated, and that the productivity that can be achieved with membranes is 10 to 20 times higher depending on the initial BSA concentration value, and with monoliths it is approximately twice that of beads, at the same superficial velocity.

scholarly journals Synthesis, Characterization, and Properties of Sulfonated Chitosan for Protein Adsorption

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Xiaomin Zhang ◽  
Jie Sun
Keyword(s):  
Ion Exchange ◽  
Protein Purification ◽  
Protein Adsorption ◽  
Protein Separation ◽  
Binding Capacity ◽  
Adsorption Properties ◽  
Synthesis Process ◽  
Exchange Adsorption ◽  
Good Potential

Chitosan sulfate was prepared and characterized as a new chromatography media for protein separation. The degree of sulfonation of chitosan could be well controlled and impacted under conditions in the synthesis process. The prepared chitosan sulfate shows improved binding capacity with proteins. Sulfonated chitosan shows improved ion-exchange adsorption properties with proteins, which could have good potential in protein purification.

scholarly journals A simple magnetic separation method for high-yield isolation of pure primary microglia

2011 ◽  
Vol 194 (2) ◽  
pp. 287-296 ◽  
Author(s):  
Richard Gordon ◽  
Colleen E. Hogan ◽  
Matthew L. Neal ◽  
Vellareddy Anantharam ◽  
Anumantha G. Kanthasamy ◽  
...  
Keyword(s):  
Magnetic Separation ◽  
Separation Method ◽  
High Yield ◽  
Primary Microglia
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