Increase in IgG-binding Capacity of Recombinant Protein a Immobilized on Heterofunctional Amino and Epoxy Agarose

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

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Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification

Applied and Environmental Microbiology ◽

10.1128/aem.01014-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7394-7397 ◽

Cited By ~ 47

Author(s):

Jane A. Brockelbank ◽

Verena Peters ◽

Bernd H. A. Rehm

Keyword(s):

Escherichia Coli ◽

Immunoglobulin G ◽

Binding Capacity ◽

Protein A ◽

Coli Strain ◽

Pha Synthase ◽

Enzyme Linked Immunosorbent Assay ◽

Ionization Time ◽

Flight Mass Spectrometry ◽

Igg Binding

ABSTRACT The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.

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Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen

Reproduction ◽

10.1530/reprod/120.1.19 ◽

2000 ◽

pp. 19-32 ◽

Cited By ~ 19

Author(s):

ML Martinez ◽

JD Harris

Keyword(s):

Menstrual Cycle ◽

Recombinant Protein ◽

Antibody Titre ◽

Zona Pellucida ◽

Protein A ◽

Papio Cynocephalus ◽

Cynomolgus Monkeys ◽

Menstrual Cycles ◽

Normal Menstrual Cycle

Immunization of female mammals with native zona pellucida (ZP) proteins is known to cause infertility. Since each human ZP protein is now available as a purified recombinant protein, is it possible to compare the immunocontraceptive potential of each ZP protein. A breeding study was conducted in cynomolgus monkeys (Macaca fasicularis) after immunization with recombinant human ZP (rhZP) proteins (ZPA, ZPB, ZPC) separately and in combinations. This study demonstrated that immunization with recombinant human ZPB (rhZPB) protein caused cynomolgus monkeys to become infertile for 9-35 months. A second study was conducted in baboons (Papio cynocephalus), which yielded a similar result. The baboons immunized with rhZPB became infertile for 9 to > 20 months. During the time of maximum antibody titre, some animals experienced disruption of the menstrual cycle, but eventually all of the animals resumed normal menstrual cycles. Control animals and animals immunized with other rhZP proteins all became pregnant before any of the rhZPB-treated animals. This is the first study in which a recombinant ZP protein has consistently induced infertility in a primate without permanent disruption of the normal menstrual cycle.

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

ACS Applied Nano Materials ◽

10.1021/acsanm.1c00487 ◽

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

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Biophysical and formulation studies of theSchistosoma mansoniTSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.25788 ◽

2013 ◽

Vol 9 (11) ◽

pp. 2351-2361 ◽

Cited By ~ 9

Author(s):

Weiqiang Cheng ◽

Elena Curti ◽

Wanderson C Rezende ◽

Clifford Kwityn ◽

Bin Zhan ◽

...

Keyword(s):

Recombinant Protein ◽

Vaccine Candidate ◽

Protein A ◽

Extracellular Domain ◽

Intestinal Schistosomiasis ◽

Vaccine Candidate Antigen ◽

Candidate Antigen

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DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein

Journal of Medical Microbiology ◽

10.1099/jmm.0.46217-0 ◽

2006 ◽

Vol 55 (4) ◽

pp. 375-378 ◽

Cited By ~ 14

Author(s):

Daniela M. Ferreira ◽

Eliane N. Miyaji ◽

Maria Leonor S. Oliveira ◽

Michelle Darrieux ◽

Ana Paula M. Arêas ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Recombinant Protein ◽

Dna Vaccines ◽

Protein A ◽

Surface Protein ◽

Humoral Response ◽

Cost Effective ◽

Promising Candidate ◽

Pneumococcal Surface Protein A ◽

Antibody Levels

Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.

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Bordetella bronchiseptica Adherence to Cilia Is Mediated by Multiple Adhesin Factors and Blocked by Surfactant Protein A

Infection and Immunity ◽

10.1128/iai.73.6.3618-3626.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3618-3626 ◽

Cited By ~ 29

Author(s):

Jessica A. Edwards ◽

Nathan A. Groathouse ◽

Scott Boitano

Keyword(s):

Binding Assay ◽

Binding Capacity ◽

Protein A ◽

Upper Airway ◽

Surfactant Protein ◽

Bordetella Bronchiseptica ◽

Surfactant Protein A ◽

Dermonecrotic Toxin ◽

Bacterial Adhesins ◽

Tracheal Epithelial

ABSTRACT In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg− phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg− strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.

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