scholarly journals Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

2019 ◽  
Vol 20 (1) ◽  
pp. 161 ◽  
Author(s):  
Tuhidul Islam ◽  
Amith D. Naik ◽  
Yasuhiro Hashimoto ◽  
Stefano Menegatti ◽  
Ruben G. Carbonell
Keyword(s):  
Binding Capacity ◽  
Protein A ◽  
Chinese Hamster ◽  
High Capacity ◽  
Alkaline Solutions ◽  
Peptide Ligand ◽  
Dynamic Binding ◽  
Ligand Density ◽  
Host Cell Proteins ◽  
Igg Binding

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.

scholarly journals Focused peptide library screening as a route to a superior affinity ligand for antibody purification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krištof Bozovičar ◽  
Barbara Jenko Bizjan ◽  
Anže Meden ◽  
Jernej Kovač ◽  
Tomaž Bratkovič
Keyword(s):  
Binding Capacity ◽  
Protein A ◽  
Downstream Processing ◽  
Phage Display Library ◽  
Enrichment Ratio ◽  
Peptide Ligand ◽  
Staphylococcal Protein A ◽  
Dynamic Binding ◽  
Linear Peptide

AbstractAffinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.

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

2006 ◽  
Vol 72 (11) ◽  
pp. 7394-7397 ◽  
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.

scholarly journals Multiplex secretome engineering enhances recombinant protein production and purity

2019 ◽  
Author(s):  
Stefan Kol ◽  
Daniel Ley ◽  
Tune Wulff ◽  
Marianne Decker ◽  
Johnny Arnsdorf ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Production ◽  
Protein A ◽  
Substantial Reduction ◽  
Chinese Hamster ◽  
Exchange Chromatography ◽  
Cell Protein ◽  
Metabolic Demand ◽  
Cho Cell ◽  
Host Cell Proteins

AbstractHost cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

scholarly journals Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery

2019 ◽  
Vol 20 (7) ◽  
pp. 1729 ◽  
Author(s):  
R. Lavoie ◽  
Alice di Fazio ◽  
R. Blackburn ◽  
Michael Goshe ◽  
Ruben Carbonell ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Mixed Mode ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Peptide Ligand ◽  
Host Cell Proteins ◽  
Ligand Discovery ◽  
Exchange Resins

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.

scholarly journals Ion exchange chromatography of monoclonal antibodies: Effect of resin ligand density on dynamic binding capacity

2009 ◽  
Vol 1216 (20) ◽  
pp. 4366-4371 ◽  
Author(s):  
Ann Marie Hardin ◽  
Chithkala Harinarayan ◽  
Gunnar Malmquist ◽  
Andreas Axén ◽  
Robert van Reis
Keyword(s):  
Monoclonal Antibodies ◽  
Ion Exchange ◽  
Binding Capacity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dynamic Binding ◽  
Ligand Density ◽  
Dynamic Binding Capacity

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis

2019 ◽  
Vol 117 (2) ◽  
pp. 438-452 ◽  
Author(s):  
R. Ashton Lavoie ◽  
Alice Fazio ◽  
Taufika Islam Williams ◽  
Ruben Carbonell ◽  
Stefano Menegatti
Keyword(s):  
Ligand Binding ◽  
Host Cell ◽  
Chinese Hamster Ovary ◽  
Proteomic Analysis ◽  
Chinese Hamster ◽  
Peptide Ligand ◽  
Host Cell Proteins ◽  
Targeted Capture

scholarly journals Nonwoven Ion-Exchange Membranes with High Protein Binding Capacity for Bioseparations

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 181
Author(s):  
Solomon Mengistu Lemma ◽  
Cristiana Boi ◽  
Ruben G. Carbonell
Keyword(s):  
Ion Exchange ◽  
Anion Exchange ◽  
Binding Capacity ◽  
Dynamic Binding ◽  
Host Cell Proteins ◽  
High Protein Binding ◽  
Protein Binding Capacity ◽  
Flow Through ◽  
Exchange Membrane ◽  
Culture Supernatants

This study presents the preparation and characterization of UV-grafted polybutylene terepthalate (PBT) ion exchange nonwoven membranes for chromatographic purification of biomolecules. The PBT nonwoven was functionalized with sulfonate and secondary amine for cation and anion exchange (CEX and AEX), respectively. The anion exchange membrane showed an equilibrium static binding capacity of 1300 mg BSA/g of membrane, while the cationic membranes achieved a maximum equilibrium binding capacity of over 700 mg hIgG/g of membrane. The CEX and AEX membranes resulted in dynamic binding capacities under flow conditions, with a residence time of 0.1 min, of 200 mg hIgG/mL of membrane and 55 mg BSA/mL of membrane, respectively. The selectivity of the PBT-CEX membranes was demonstrated by purifying antibodies and antibody fragments (hIgG and scFv) from CHO cell culture supernatants in a bind-an-elute mode. The purity of the eluted samples exceeded 97%, with good log removal values (LRV) for both host cell proteins (HCPs) and DNA. The PBT-AEX nonwoven membranes exhibited a DNA LRV of 2.6 from hIgG solutions in a flow-through mode with little loss of product. These results indicate that these membranes have significant potential for use in downstream purification of biologics.

In Vitro Removal of Human Igg by Pseudobiospecific Affinity Membrane Filtration on a Large Scale. A Preliminary Report

1995 ◽  
Vol 18 (7) ◽  
pp. 392-398 ◽  
Author(s):  
S.M.A. Bueno ◽  
K. Haupt ◽  
M.A. Vijayalakshmi
Keyword(s):  
Membrane Filtration ◽  
Large Scale ◽  
Binding Capacity ◽  
Protein A ◽  
High Capacity ◽  
Ether Linkage ◽  
Human Igg ◽  
Affinity Membrane ◽  
Affinity Membranes

We have developed a pseudobiospecific affinity membrane device for selective removal of human IgG from plasma or serum in vitro for clinical apheresis application. The pseudobiospecific affinity ligand L-histidine was immobilized through an ether linkage onto poly(ethylenevinyl alcohol) hollow fiber cartridge. The obtained affinity membranes showed high selectivity for IgG adsorption from untreated human serum. These membranes are able to adsorb lgG1, lgG2, lgG3 if Mops buffer is used, and more selectively lgG1 and lgG3 in Tris-HCl buffer. With respect to the binding capacity, the pseudobiospecific affinity membrane used showed a higher capacity as compared to protein A-membranes described in the literature. Due to the high capacity, specificity and stability of the histidine affinity membranes, in addition to their lower cost, the approach proposed in this paper may offer a useful alternative to protein A based devices in the treatment of immune-related diseases.

scholarly journals Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity

2014 ◽  
Vol 1362 ◽  
pp. 180-185 ◽  
Author(s):  
Timothy M. Pabst ◽  
Ronnie Palmgren ◽  
Annika Forss ◽  
Jelena Vasic ◽  
Mariko Fonseca ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Dynamic Binding ◽  
Staphylococcal Protein ◽  
Dynamic Binding Capacity ◽  
High Dynamic
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